Transcriptional control signals required for tumor (T)-antigen trans-activation of the simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the trans-acting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.