SV40 large T antigen interacts specifically with three different DNA sequences within the SV40 control region namely site I, II and III, in order to regulate the viral DNA replication and the stimulation of late, or repression of early viral transcription. We define three different plasmids, containing either site I alone, site II with SV40 specific flanking sequences or both sites I and II with additional neighbouring sequences which were used to study the interaction of T antigen with each of its binding sites. We found that binding of T antigen to site II reduced binding of T antigen to site I as opposed to the interaction of T antigen with site I alone. These results were supported by our findings that T antigen from an SV40 temperature-sensitive mutant, tsA58, which is heat-sensitive for binding to site II, exhibited the same binding affinity for site I as for the DNA fragment containing both binding sites. Thus, binding of T antigen to binding site II seems to interfere with binding to site I, thereby influencing the regulatory functions of T antigen in the repression of early viral gene transcription.