Integrin alphavbeta3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin alphavbeta3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin alphavbeta3-positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate) as a new radiotracer for imaging breast cancer integrin alphavbeta3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 = 51 +/- 11 nM) was slightly higher than that of its dimeric analogue (IC50 = 78 +/- 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 = 55 +/- 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 +/- 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 +/- 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 +/- 0.87 %ID/g and 7.30 +/- 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin alphavbeta3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only approximately 15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin alphavbeta3-positive tumors by SPECT.