Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv(4)-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a monobiotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv(4)-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents, good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-trigalactose blood clearance reagent, 2, was designed, synthesized, and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g., (111)In, (90)Y, (177)Lu) could be used in the pretargeting protocols employing scFv(4)-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [(125)I]scFv(4)-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 and [(111)In]4b) with standard reagents (1 and [(111)In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv(4)-SAv-S45A or scFv(4)-SAv-Y43A) were employed in combination with CA 2 and [(111)In]4b. Importantly, normal tissue concentrations of [(111)In]4b were similar to those obtained using the standard reagents (1 and [(111)In]3b), except that the blood and liver concentrations were slightly higher with the new reagents. While the reasons for the higher blood and liver concentrations are unknown, the differences in the galactose structures of the clearance agents 1 and 2 may play a role.