The metabolism of (177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-(4-aminobenzoyl)-QWAVGHLM-NH(2)), a radiotherapeutic compound in clinical development that binds to GRP and NMB receptors, was studied in vitro (mouse, rat and human plasma, mouse kidney homogenate) and in vivo (by analysis of mouse and rat plasma and urine following IV injection of (177)Lu-AMBA). The primary metabolites were Lu-DO3A-CH(2)CO-G-Abz4-R, where R = -Q-OH (A), -QW-OH (B), and -QWAVGH-OH (C). Minor amounts of (D) where R = -QWAVGHLM-OH and (E) -QWAVGHL-OH were also observed. Clearance of (177)Lu-AMBA and of radioactivity from mouse and rat blood was rapid in vivo. In mouse and rat urine, only metabolites Lu-A and Lu-B were found-no parent drug was excreted. Unmetalated ligands and (nat)Lu and (177)Lu complexes for Lu-AMBA metabolites A-E were synthesized, characterized by HPLC and MS, and used to perform in vitro competition and direct binding studies on GRP receptor-positive PC-3 (human prostate) cancer cells. Biodistribution studies with (177)Lu-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact (177)Lu-AMBA. IC(50) values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against (177)Lu-AMBA. No direct binding to PC-3 cells was observed with (177)Lu-labeled A-C, confirming IC(50) results. (177)Lu-labeled metabolites A-E showed no uptake in GRP-receptor positive tumor or pancreas in PC-3 tumor bearing mice. All metabolites were rapidly excreted via the renal route (approximately 78-87%) within 1 h. These results demonstrate that the tumor uptake observed with (177)Lu-AMBA is due to parent drug and not due to any of its identified metabolites.