Deletion and substitution mutations in the control region of simian virus 40 (SV40) were used to study regulation of early transcription by T antigen. A mutant, pIN4, containing substitutions within T antigen binding site II, is transcribed in a cell-free extract at wild-type efficiency but is unable to be repressed in vitro by purified T antigen under conditions that fully repress wild-type transcription. These results suggest a functional role for T antigen binding site II in the repression of early SV40 transcription. To investigate autoregulation in vivo, transcription from the SV40 early promoter was quantitated in COS7 monkey cells transfected with plasmid vectors carrying the mouse dihydrofolate reductase gene (SV-dhfr vectors). Mutant SV-dhfr vectors lacking T antigen site I or site II sequences overproduce dhfr RNA from the SV40 early promoter three to four fold, whereas deletion of both sites I and II or the presence of a temperature-sensitive T antigen (tsA209) results in an eight to ten fold increase in dhfr RNA. Our results indicate that binding of T antigen to both sites I and II plays a role in autoregulation.