Diagnostic and therapeutic procedures utilizing the high affinity streptavidin (SA)/biotin system are being investigated for in vivo use. We are developing a rapid two-step imaging technique for the diagnosis of deep venous thrombosis and pulmonary embolism. The optimal SA-bound targeting moiety would circulate adequately for sufficient lesion accumulation, but nonbound reagent would clear in a reasonably short time before the injection of radiolabeled biotin. The objective of this study was to cross-link SA and galactose-modified SA to GC4 antifibrin monoclonal antibody and to study the pharmacokinetics and biodistribution of the radiolabeled GC4-SA conjugates after injection into rabbits. A cross-linking method was developed for the synthesis of the GC4-SA conjugates via the addition reaction of sulfhydryl containing SA derivatives with maleimide-GC4. In vivo, radiolabeled trigalactose modified SA-GC4 exhibited a much faster blood clearance compared to mono-galactose modified GC4-SA or GC4-SA containing no galactose.