Pretargeting methodologies can produce high tumor:blood ratios, but their role in cancer radioimmunotherapy (RAIT) is uncertain. A pretargeting method was developed using a streptavidin (StAv) conjugate of MN-14 IgG, an anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (mab) as the primary targeting agent, an anti-idiotype antibody (WI2 IgG) as a clearing agent, and DTPA- or DOTA-conjugated biotin as the radiolabeled targeting agent. A variety of reagents and conditions were examined to optimize this method. At 3 h, 111In-DTPA-peptide-biotin tumor uptake was 3.9 +/- 0.8% per gram and tumor:blood ratios were > 11:1. By 24 h, this ratio was 178:1, but tumor accretion declined in accordance with the gradual loss of StAv-MN-14 from the tumor. Tissue retention was highest in the liver and kidneys, but their tumor:organ ratios were > 2:1. Dosimetry predicted that radiolabeled MN-14 alone would deliver higher tumor doses than this pretargeting method. Increasing the specific activity and using DOTA-biotin in place of DTPA increased tumor uptake nearly 2-fold, but analysis of StAv-MN-14's biotin-binding capacity indicated over 90% of the initial biotin-binding sites were blocked within 24 h. Animals fed a biotin-deficient diet had 2-fold higher 111In-DOTA-biotin uptake in the tumor, but higher uptake also was observed in all normal tissues. Although exceptionally adept at achieving high tumor:blood ratios rapidly, the tumor uptake of radiolabeled biotin with this pretargeting method is significantly (p < 0.0001) lower than that with a radiolabeled antibody. Endogenous biotin and enhanced liver and kidney uptake may limit the application of this method to RAIT, especially when evaluating the method in animals, but with strategies to overcome these limitations, this pretargeting method could be an effective therapeutic alternative.