Integrins are transmembrane glycoproteins with two noncovalently bound α and β subunits. The two subunits mediate cell–cell and cell–extracellular matrix (ECM) interactions, and they act downstream of several primary signaling events that lead to angiogenesis (1-4). Integrins comprise a large family of cell adhesion molecules, and integrin αβ appears to be the most attractive member for angiogenesis-targeted imaging and therapy because of its critical involvement in tumor angiogenesis, development, and metastasis. Integrin αβ is minimally expressed in normal blood vessels but is significantly upregulated in newly sprouting vasculature in tumors (2, 4, 5). A significant effort has been made to generate various imaging agents targeting integrin αβ (1, 4, 6-9). Generally, these agents can be categorized as antibodies, peptides, small-molecule peptidomimetics, and targeted nanoparticles. Vitaxin is the representative of monoclonal antibodies against integrin αβ. Vitaxin is a humanized antibody composed of human IgG-1, kappa, and the complement domain regions of the murine antibody LM 609. Regardless of its efficacy in inhibiting angiogenesis, imaging with Tc-labeled Vitaxin failed to show the tumor angiogenesis in the clinical settings because of its poor stability and rapid plasma clearance with low doses (10). Interestingly, Gutheil et al. have reported that second-generation Vitaxin-2–conjugated, gadolinium-encapsulated nanoparticles could provide enhanced and detailed imaging of rabbit carcinomas and imaging of angiogenic “hot spots” that are not seen with standard magnetic resonance imaging (11). The major limitations of monoclonal antibodies include large molecular size, low production yield, incomplete tumor penetration, and immunogenicity to host. Numerous small peptides have been identified to specifically interact with tumor neovasculature, including arginine-glycine-aspartic acid (RGD), asparagine-glycine-arginine, histidine-tryptophan-glycine-phenylalanine, and arginine-arginine-leucine (RRL) (7, 12-16). RGD tripeptide sequence is known as a cell recognition site for adhesive proteins present in the ECM and in blood. Integrin αβ binds ECM proteins through the exposed RGD tripeptide in their ligands. Both linear and cyclic RGD peptide agents have been proven to be useful in imaging tumor neovasculature. In general, linear peptides are broken down rapidly and occupy a wide range of conformations, resulting in low binding affinity and less specific accumulation within tumors. Short cyclic peptides are superior to linear peptides for their pharmacokinetics due to the fact that they are trapped in the active conformation and are more resistant to proteolysis. The pharmacokinetic behaviors of RGD peptides can be further improved with introduction of sugar moiety, the dimeric format of RGD peptides, or coupling with 1,4,7,10-tetraazacyclododecane--tetraacetic acid or polyethylene glycol (12, 13, 15). However, RGD peptides are less selective (binding with 8 of the 24 integrins), and their binding affinities are relatively low (50% inhibition concentration (IC), 20–70 nmol) compared to antibodies. A series of non-peptide αβ chemical antagonists including IntegriSense (IC, 4.1 nmol; binding with αβ/αβ only) have recently been shown to accumulate specifically in αβ/αβ-expressing tumors with high affinity and specificity (14). More recently, several classes of peptidomimetic integrin αβ antagonists have been reported (6, 17). These peptidomimetic antagonists consist of a rigid core scaffold bearing basic and acidic groups that mimic the guanidine and carboxylate groups of the RGD sequence. Additionally, ligand array of integrin antagonists on nanoparticles has been proven to be a viable strategy to target vascular surface receptors on endothelial cells. Imaging studies are in progress with these new strategies (6). Nevertheless, small peptides have distinct advantages such as easy synthesis, less immunogenicity, and rapid plasma clearance. In addition to RGD, the tripeptide sequence RRL has been investigated to generate imaging agents (16, 18). RRL is a tumor endothelial cell–specific binding peptide previously identified by Brown et al. using an bacterial peptide display library panned against tumor cells (19). Weller et al. have shown that the microbubble-linked RRL peptide preferentially adheres to tumor normal vasculature, and this selective adherence can be detected with ultrasound (18). Yu et al. studied the radionuclide-labeled RRL peptide (designated WT1040) for tumor imaging and demonstrated that the I-labeled RRL peptide allowed non-invasive visualization of tumors (16).