TROP-2 is a pancarcinoma marker that is expressed at high levels in many epithelial cancers, including prostate cancer (PC). The trivalent bispecific antibody TF12 (anti-TROP2 × anti-HSG [histamine-succinyl-glycine]) has shown to effectively target PC. In this study, the efficacy of pretargeted radioimmunotherapy (PRIT) with multiple cycles of TF12 and (177)Lu-labeled diHSG-peptide (IMP288) in mice with s.c. PC3 tumors was investigated and compared with that of conventional RIT with (177)Lu-labeled anti-TROP-2 mAb hRS7. The potential of one, two, and three cycles of PRIT using the TF12 pretargeted (177)Lu-IMP288 (41 MBq per cycle) was determined in mice with s.c. PC3 tumors, and compared with the efficacy and toxicity of RIT with (177)Lu-hRS7 dosed at the maximum tolerated dose (11 MBq). PRIT of two and three cycles showed significantly higher median survival (> 150 days) compared with PRIT of one cycle of TF12 and (177)Lu-IMP288 (111 days, p < 0.001) or the controls (76 days, p < 0.0001). All mice treated with the mAb (177)Lu-hRS7 survived at the end of the experiment (150 days), compared with 80% in the mice that were treated with three cycles of PRIT and 70% in the group that received two cycles of PRIT. Clinically significant hematologic toxicity was found only in the groups that received either three cycles of PRIT (p < 0.0009) or RIT (p < 0.0001). TROP-2-expressing PC can be targeted efficiently with TF12 and radiolabeled IMP288. (177)Lu-IMP288 accumulated rapidly in the tumors. PRIT of multiple cycles inhibited the growth of s.c. PC3 tumors. Clinically relevant hematological toxicity was observed in the group that received three cycles of PRIT; however, conventional RIT with the parent mAb (177)Lu-hRS7 was at least as effective with similar toxicity.