Purpose: This study examined a pretarget radioimmunotherapy strategy for treatment of an i.p. tumor model (LS174T). Experimental Design: The strategy used regional administration (i.p.) of a novel targeting molecule composed of four CC49 anti - tumor-associated glycoprotein 72 (TAG-72) single-chain antibodies linked to streptavidin as a fusion protein (CC49 fusion protein); 24 hours later, a synthetic clearing agent was administered i.v. to produce hepatic clearance of unbound CC49 fusion protein/synthetic clearing agent complexes. Four hours later, a low molecular weight radiolabeled reagent composed of biotin conjugated to the chelating agent 7,10-tetra-azacyclododecane-N,N′, N″,N‴-tetraacetic acid (DOTA) complexed with 111In-, 90Y-, or 177Lu-DOTA-biotin was injected. Results: Radiolocalization to tumor sites was superior with i.p. administration of radiolabeled DOTA-biotin as compared with i.v. administration. Imaging and biodistribution studies showed excellent tumor localization of radioactivity with 111In- or 177Lu-DOTA-biotin. Tumor localization of 111In-DOTA-biotin was 43% ID/g and 44% ID/g at 4 and 24 hours with the highest normal tissue localization in the kidney with 6% ID/g at 48 and 72 hours. Therapy studies with 90Y-DOTA-biotin at doses of 400 to 600 μCi or 177Lu-DOTA-biotin at doses of 600 to 800 μC produced significant prolongation of survival compared with controls (P = 0.03 and P < 0.01). Conclusions: Pretarget radioimmunotherapy using regional administration of CC49 fusion protein and i.p. 90Y- or 177Lu-DOTA-biotin represents a successful therapeutic strategy in the LS174T i.p. tumor model and this strategy may be applicable to human trials in patients with i.p. ovarian cancer. © 2005 American Association for Cancer Research.