The present study describes the synthesis and biological evaluation of (111)In(DOTA-3P-RGD(2)) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2); PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), (111)In(DTPA-3P-RGD(2)) (DTPA = diethylenetriaminepentaacetic acid) and (111)In(DTPA-Bn-3P-RGD(2)) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α(v)β(3) expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the (111)In-labeled 3P-RGD(2). IC(50) values of DOTA-3P-RGD(2), DTPA-3P-RGD(2) and DTPA-Bn-3P-RGD(2) were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against (125)I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting (111)InCl(3) with the RGD peptide conjugates in NH(4)OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD(2), successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15-20 min. For DTPA-3P-RGD(2) and DTPA-Bn-3P-RGD(2), the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for (111)In(DOTA-3P-RGD(2)) and ~200 mCi/mg (or ~400 mCi/μmol) for (111)In(DTPA-3P-RGD(2)). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both (111)In(DTPA-3P-RGD(2)) and (111)In(DTPA-Bn-3P-RGD(2)) showed a much faster tumor washout and poorer T/B ratios than (111)In(DOTA-3P-RGD(2)). The tumor uptake of (111)In(DOTA-3P-RGD(2)) is integrin α(v)β(3)- and RGD-specific. (111)In(DOTA-3P-RGD(2)) is metabolically stable while only ~25% of (111)In(DTPA-Bn-3P-RGD(2)) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that (111)In(DTPA-3P-RGD(2)) can be an effective integrin α(v)β(3)-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.