Tc-Labeled acetylated (Ac) dendrimer poly(amido)-amine (PAMAM) generation 5 (G5)-PEGylated folic acid (FA)-2-(-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid (1B4M DTPA) conjugate, abbreviated as Tc-G5-Ac-pegFA-DTPA, was synthesized by Zhang et al. for folate receptor (FR)-targeted imaging of FR-positive tumors (1, 2). Folate is an essential vitamin for cell synthesis of nucleotide bases. Some unique features of the FR-folate system make it extremely valuable for developing FR-targeted imaging and therapeutic agents (3-5). First, FR has a high affinity for the exogenous folate conjugates ( = ~100 pM), but they are inaccessible for the conjugates in most normal tissues. Second, FR-α isoform is overexpressed on ~40% of human cancers, where it is completely accessible to folate conjugates. Third, folate conjugates are taken up by cancer cells FR-mediated endocytosis, and FR recycles actively to the cell surface with a frequency of 5.7–20 h depending on cell types. Fourth, conjugation of imaging labels the γ-carboxyl group of folate has no apparent effects on the ligand-binding affinity to FR. Furthermore, folate is a small molecule (MW = ~441) that exhibits rapid and complete penetration of solid tumors and rapid clearance from FR-negative tissues (, <10 min). One disadvantage of the FR-folate system is that FR expresses at a relatively low density on some tumor cell surface (1–3 million FR/cell), which means that the FR-folate binding can be saturated rapidly and the imaging contrast will be limited (3, 4, 6). Sensitive imaging techniques such as positron emission tomography and single-photon emission computed tomography (SPECT) are more desirable for FR-targeted imaging (3, 4, 7). To date, a large set of folate-based radiopharmaceutical agents have been synthesized for nuclear imaging (8). In general, a chelating agent is necessary to bridge the radiolabels and the folate molecule. The chelating agent is also critical for optimizing the molecular properties of conjugates (1, 2, 9). Dendrimers represent a unique class of nanostructures that are synthesized from branched monomers in a step-wise manner. The molecular properties of dendrimers can be precisely controlled by choosing different branching monomers and surface functional groups (1, 2, 9). PAMAM is one of the most extensively studied dendrimers. Zhang et al. synthesized two folate-based compounds, Tc-G5-Ac-pegFA-DTPA and Tc-G5-Ac-FA-DTPA, and one control, Tc-G5-Ac-DTPA, with PAMAM G5 dendrimer and folate (1, 2). To increase the solubility and decrease the nonspecific cellular uptake of the agents, the primary amines on the surface of PAMAM dendrimers were partially converted to acetamide moieties, which were used to link with the bifunctional chelating agent 1B4M DTPA. Folate was either PEGylated or left unPEGylated, and its c-carboxyl group was used to conjugate with the primary amine of PAMAM. The two folate-based conjugates exhibited excellent stability, rapid clearance from blood, and high accumulation in tumor xenografts (1, 2). This chapter describes the results obtained with Tc-G5-Ac-pegFA-DTPA. Another chapter in MICAD describes the data obtained with Tc-G5-Ac-FA-DTPA.