I have mapped the regions of simian virus 40 (SV40)-encoded large tumor (T) antigen that remained associated with origin-containing SV40 DNA after digestion with various concentrations of Pronase E, a nonspecific protease. Immunoaffinity-purified, labeled T antigen was bound to SV40 DNA-cellulose and treated with Pronase E. A "core" region representing amino acids 140 to about 281 was identified by peptide mapping in the fraction that remained bound to the DNA-cellulose after treatment with a high (135 micrograms/ml) concentration of enzyme. This region corresponds to the DNA-binding domain of the protein molecule. After treatment with Pronase E at 66 micrograms/ml, the bound fraction consisted of the DNA-binding domain and a region that extends to residue 371. This larger protein segment binds more stably to the viral DNA than does the core by itself. At lower concentrations of Pronase E, additional sequences from the NH2-terminal region of T antigen and from the COOH-half of the molecule were observed in the bound fractions. Linear maps of resistant regions, generated for each concentration of protease used, provide information about the geometry of the protein molecule associated with the DNA. I suggest that regions that are easily cleaved by the protease are exposed in the DNA-protein complex, whereas those that remain bound to the DNA at increasing concentrations of the enzyme represent segments that are in progressively closer proximity to the viral DNA origin.