Integrin α_vβ₃ plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of α_vβ₃ expression and targeted radionuclide therapy. In this study, we developed ⁶⁴Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)]₂}₂ (RGD tetramer) and E(E{E[c(RGDyK)]₂}₂)₂ (RGD octamer), for PET imaging of tumor integrin α_vβ₃ expression. Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), the peptides were labeled with ⁶⁴Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the α_vβ₃-binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. RGD octamer had significantly higher integrin α_vβ₃-binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). ⁶⁴Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than ⁶⁴Cu-DOTA-RGD tetramer in both tumor models tested. The integrin α_vβ₃ specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of ⁶⁴Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin α_vβ₃-binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin α_vβ₃.