Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a new class of radiotracers with potential for early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response in cancer patients. This article describes the synthesis of two cyclic RGD peptide dimer conjugates, DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2) (DOTA-3PEG(4)-dimer: DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and DOTA-G(3)-E[G(3)-c(RGDfK)](2) (DOTA-3G(3)-dimer: G(3) = Gly-Gly-Gly). Integrin alpha(v)beta(3) binding affinities of cyclic RGD peptides were determined by competitive displacement of (125)I-echistatin bound to U87MG human glioma cells and follow the order of DOTA-E{E[c(RGDfK)](2)}(2) (DOTA-tetramer: IC(50) = 10 +/- 2 nM) > DOTA-3G(3)-dimer (IC(50) = 62 +/- 6 nM) approximately DOTA-3PEG(4)-dimer (IC(50) = 74 +/- 3 nM) > DOTA-E[c(RGDfK)](2) (DOTA-dimer: IC(50) = 102 +/- 5 nM). The addition of PEG(4) and G(3) linkers between two cyclic RGD motifs in DOTA-3G(3)-dimer and DOTA-3PEG(4)-dimer makes it possible for them to achieve the simultaneous integrin alpha(v)beta(3) binding in a bivalent fashion. Both (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3G(3)-dimer) were prepared in high yield with specific activity being >50 Ci/mmol. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG human glioma xenografts. The results from those studies show that PEG(4) and G(3) linkers are particularly useful for improving tumor uptake and clearance kinetics of (64)Cu radiotracers from the nontumor organs, such as kidneys, liver, and lungs. There is a linear relationship between the tumor size and %ID tumor uptake, suggesting that (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3PEG(4)-dimer) might be useful for noninvasive monitoring of tumor growth or shrinkage during antiangiogenic therapy. MicroPET imaging data clearly demonstrate the utility of (64)Cu(DOTA-3G(3)-dimer) as a new PET radiotracer for imaging integrin alpha(v)beta(3)-positive tumors.