We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI.