The purpose of this study was to test the three-step targeting of tumors in mice using biotinylated antibody, streptavidin and radiolabeled biotin. Nude mice bearing subcutaneous LS180 human colon cancer xenografts were intravenously administered with 200 microg of the biotinylated anti-Tn monoclonal antibody MLS128, and 2 days later they got intravenous injection of 50 microg of streptavidin. They were intravenously injected 1, 4 or 7 days later with 0.5 microg of 111In-diethylenetriamine pentaacetic acid (DTPA)-biotin. The tumor uptake, determined 2 h later, was 1.4, 0.5 and 0.6% injected dose/gram of tissue (ID/g), respectively, and the blood radioactivity was 1.0, 0.2 and 0.2% ID/g, respectively. When the interval between the streptavidin and radiolabeled biotin injections was prolonged from 1 day to 7 days, the tumor-to-blood ratio 2 h after injection of 111In-labeled biotin increased from 1.5 to 4.0. Clear tumor images were obtained as early as 2 h after injection of radiolabeled biotin. In conclusion, these preliminary data suggested that the three-step method using the streptavidin-biotin system would be applicable in an experimental mouse tumor model and provides images of tumors rapidly and clearly after injection of radiolabeled biotin.