BACKGROUND: Prostate cancer is radiosensitive. Prostate-specific membrane antigen (PSMA) is selectively overexpressed on advanced, castration-resistant tumors. Lutetium-177-labeled anti-PSMA monoclonal antibody J591 ( Lu-J591) targets prostate cancer with efficacy and dose-response/toxicity data when delivered as a single dose. Dose fractionation may allow higher doses to be administered safely. METHOD: Men with metastatic castration-resistant prostate cancer refractory to or refusing standard treatment options with normal neutrophil and platelet counts were enrolled in initial phase 1b dose-escalation cohorts followed by phase 2a cohorts treated at recommended phase 2 doses (RP2Ds) comprising 2 fractionated doses of Lu-J591 2 weeks apart. Lu-J591 imaging was performed after treatment, but no selection for PSMA expression was performed before enrollment. Phase 2 patients had circulating tumor cell (CTC) counts assessed before and after treatment. RESULTS: Forty-nine men received fractionated doses of Lu-J591 ranging from 20 to 45 mCi/m ×2 two weeks apart. The dose-limiting toxicity in phase 1 was neutropenia. The RP2Ds were 40 mCi/m and 45 mCi/m ×2. At the highest RP2D (45 mCi/m ×2), 35.3% of patients had reversible grade 4 neutropenia, and 58.8% of patients had thrombocytopenia. This dose showed a greater decrease in prostate-specific antigen (PSA) levels and longer survival (87.5% with any PSA decrease, 58.8% with >30% decrease, 29.4% with >50% decrease; median survival, 42.3 months [95% confidence interval, 19.9-64.7]). Fourteen of 17 (82%) patients with detectable CTCs experienced a decrease in CTC count. Overall, 79.6% of patients had positive PSMA imaging; those with less intense PSMA imaging tended to have poorer responses. CONCLUSION: Fractionated administration of Lu-J591 allowed higher cumulative radiation dosing. The frequency and depth of PSA decrease, overall survival, and toxicity (dose-limiting myelosuppression) increased with higher doses.