One pretargeting approach to cancer radioimmunotherapy utilizes an antibody-streptavidin conjugate that is first localized to the tumor. A "clearing agent" is then administered to remove the excess bioconjugate from blood, followed by injection of the radiolabeled biotin therapeutic. In this study, the role of streptavidin-biotin affinity in this pretargeting system was investigated for the first time in vivo, with a reduced affinity, site-directed streptavidin mutant and with radiolabeled bis-biotin reagents. The S45A streptavidin mutant (SA-S45A), which displays a faster off-rate for biotin, was utilized with a bivalent biotin carrier that retains high avidity for the streptavidin mutant. Mice were fed either a normal or biotin-deficient diet, yielding serum endogenous biotin concentrations of 31 nM and 5 nM, respectively. Lymphoma-bearing nude mice pretargeted with 1F5 Antibody-SA-Wild Type (WT) bioconjugates produced (125)I-bis-biotin tumor concentrations of 2.2%ID/g and 7.0%ID/g in mice fed normal diets vs biotin-deficient diets. (125)I-bis-biotin tumor concentrations of mice pretargeted with 1F5-SA-S45A were 12%ID/g and 10%ID/g for mice fed normal and biotin-deficient diets, respectively. However, poor clearance of the 1F5-SA-S45A with the biotinylated clearing agent led to high normal organ concentrations of (125)I-bis-biotin. A galactosylated human serum albumin (HSA) modified with bis-biotin was then tested, and normal organ (125)I-bis-biotin concentrations were significantly reduced. Tumor-to-organ ratios achieved for 1F5-SA-S45A with the HSA-bis-biotin clearing agent in mice with high serum biotin were similar to those achieved with 1F5-SA-WT in mice with low serum biotin. These results demonstrate that exchange of bound endogenous biotin with lower affinity streptavidin mutants is possible, and that corresponding use of bis-biotin carriers can nearly eliminate the differences in therapeutic radioactivity at the tumor site in animals on normal vs biotin-deficient diets. The results also interestingly demonstrate, however, that improved clearance agents capable of removing the lower affinity streptavidin-antibody conjugate are needed to achieve comparable specificity in tumor to blood or normal organ ratios.