Simian virus 40 tumor antigen (T-antigen) plays a central role in determining which gene is transcribed from viral DNA late in infection. Results from several studies have led to a model in which the binding of T-antigen to the viral origin of replication results in repression of transcription from the stronger early gene promoter and stimulation of transcription from the late gene promoter. We have tested this model by determining directly the occupancy of the T-antigen binding site in the origin of replication of the late transcription complex. Thus, viral transcription complexes were digested with BglI, a restriction enzyme that cuts in the viral replication origin. The enzyme cleaved 78(+/- 12)% of the late transcription complexes. Control experiments demonstrated that cleavage is blocked when T-antigen is bound to the origin site, that exogenously added T-antigen can bind to the site in the transcription complex, and that T-antigen is not released during isolation of the complex. These results indicate that most of the late transcription complexes do not have T-antigen bound to the origin site, and are therefore inconsistent with models that require this site to be occupied by T-antigen to maintain proper regulation of gene transcription late in infection.