Radiolabeled monoclonal antibodies (MoAbs) have been used for radioimmunotherapy (RIT) of human colon cancer xenografts in an attempt to develop promising clinical approaches to improving therapy success. Several strategies have been investigated to accomplish this task while decreasing toxicity. The CC49 antibody was selected for the present studies because of its relatively high affinity constant (16.2 × 109 M-1) for the high molecular weight TAG-72 mucinous antigen secreted by human colon cancer cells. In previous studies, when CC49 was labeled with 131I, it demonstrated a substantial therapeutic advantage over the lower affinity antibody (B723) reactive with TAG-72. One of the chief problems hi achieving cures with RIT is that hematological toxicity limits the quantity of radionuclide that can be administered. In other studies of dose fractionation using athymic nude mice and 131l-labeled intact MoAbs reactive with human colon cancer xenografts, multiple administrations at approximately 1-week intervals were found to produce more prolonged tumor growth inhibition and less toxicity than single administrations. The purpose of this study was to investigate the therapeutic efficacy and toxicity of 131I-labeled CC49 MoAb administered with short fractionation schedules against human colon cancer xenografts to determine the optimal treatment schedule, with the ultimate aim of applying this approach in clinical trials. The results reported here demonstrate that in an animal colon cancer xenograft model, RIT delivered in a fractionated schedule clearly presents a therapeutic advantage. For example, one administration of 600 μCi 131I-labeled CC49 to LS174T tumor-bearing mice was lethal to approximately 25% of mice but produced no tumor regressions. Fractionation of this dose to two administrations of 300 μCi 131l-labeled CC49 at a 3-day interval resulted in tumor regression in approximately 30% of the animals, accompanied by a similar 25% death rate. The administration of 300 μCi 131I-labeled CC49 at a 7-day interval resulted in 15% animal lethality, but no complete tumor regressions. When three administrations of 300 μCi 131Mabeled CC49 were given over a 1-week period on days 0, 3, and 7, tumor regressions occurred in approximately 40% of the animals, accompanied by a 30% death rate. Moreover, three administrations of 300 μCi 131I-labeled CC49 resulted in 20% tumor recurrence, whereas two administrations of 131I-labeled CC49 resulted in 60% tumor recurrence. The data in the present study concerning the concentration of 125I-labeled CC49 in LS174T tumors following one or two treatments with 131I-labeled CC49 at a 3-day interval suggest that a higher concentration of radiolabeled antibody was maintained in the tumor for a longer period of time than would have occurred with a single administration of 131I-labeled CC49. The studies reported here demonstrate that fractionated administrations of 131I-labeled CC49 at short time intervals can clearly elicit both tumor regressions and cures with limited toxicity in a model using relatively large established human colon carcinoma xenografts. The results of the present study suggest that initial trials in humans should be designed with the goal of determining a fractionation interval of a nonimmunogenic genetically engineered or human antibody that reduces bone marrow toxicity but permits similar tumor uptake of each radiolabeled antibody injection.