Recently developed techniques allow rapid and accurate determination of protein-induced DNA unwinding. We have used such techniques to determine whether SV40 antigen unwinds DNA. Assays were performed with purified wild type T antigen, D2 protein, and the SV80 T protein over a wide range of protein concentrations. Additionally, several DNA templates were used, including SV40 DNA, pBR322 DNA, and two plasmid DNAs containing one and three copies of the SV40 origin of replication. None of the purified proteins showed unwinding activity either in the presence or absence of ATP and Mg2+. As a positive control, we found that unwinding of pBR322 DNA by Escherichia coli RNA polymerase occurred under identical assay conditions. Protein-DNA interactions that involve unwinding of the helix are sensitive to the superhelical density of the DNA. Using a filter binding assay, we found that the efficiency of the binding of T antigen to the viral origin of replication does not depend upon the topological state of the DNA. This result further supports our conclusion that SV40 T antigen is not an unwinding protein.