The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of carcinomas and has been the target for detection and treatment of these neoplasms in animals. In particular, analogues of the tetradecapeptide bombesin (BN) have been radiolabeled with 99mTc and 111In for detection of GRPR-positive tumors by gamma ray scintigraphy. The goal of this study was to evaluate the potential of the bombesin analogue, DOTA-Aoc-BN(7-14), for positron-emission tomographic (PET) imaging after radiolabeling with the positron-emitter 64Cu. A saturation binding assay on PC-3 human prostate cancer cells showed that 64Cu-DOTA-Aoc-BN(7-14) had an equilibrium binding constant (Kd) of 6.1 ± 2.5 nM and a receptor concentration (Bmax) of 2.7 ± 0.6 × 105 receptors/cell. The radiolabeled analogue also showed rapid internalization with 18.2% internalized into 105 PC-3 cells by 2 h. The tumor localization of 64Cu-DOTA-Aoc-BN(7-14) was 5.5% injected dose per gram in athymic nude mice bearing PC-3 xenografts at 2 h postinjection. The tumor retention with respect to the 2 h value was 76% and 45% at 4 and 24 h, respectively, and was GRPR-mediated as shown by inhibition with a coinjection of excess peptide. MicroPET imaging of 64Cu-DOTA-Aoc-BN(7-14) in athymic nude mice bearing subcutaneous PC-3 tumors showed good tumor localization. Further studies with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) suggested that low blood flow to the PC-3 tumors may have limited the localization of 64Cu-DOTA-Aoc-BN(7-14). This study demonstrates that 64Cu-DOTA-Aoc-BN(7-14) can be used to detect GRPR-positive tumors by PET imaging.