The simian virus 40 large T antigen is a promiscuous transcriptional activator of many viral and cellular promoters. We show that the promoter structure necessary for T antigen-mediated transcriptional activation is very simple. A TATA or initiator element is required, in addition to an upstream factor-binding site, which can be quite variable. We found that promoters containing an SP1-, ATF-, AP1-, or TEF-I-binding site, in conjunction with a TATA element, can all be activated in the presence of T antigen. In addition, preference for specific TATA elements was indicated. Promoters containing the HSP70 TATA element functioned better than those with the adenovirus E2 TATA element, while promoters containing the simian virus 40 (SV40) early TATA element failed to be activated. In addition, simple promoters containing the initiator element from the terminal deoxynucleotidyltransferase gene could be activated by T antigen. The SV40 late promoter, a primary target for T antigen transcriptional activation, conforms to this simple promoter structure. The region from which most late transcripts initiate contains a cluster of initiator-like elements (SV40 nucleotides [nt] 250 to 335) forming an initiator region (IR). This lies downstream of the previously described octamer-TEF element (SV40 nt 199 to 218) which contains the TEF-I-binding sites shown to be necessary for T antigen-mediated transcriptional activation of the late promoter. We show that a simple late promoter made up of IR sequences and octamer-TEF element-containing sequences is transcriptionally activated by T antigen. These experiments also showed that specific sequences in the IR, SV40 nt 272 to 294, are particularly important for late promoter activation. Previous findings (M. C. Gruda, J. M. Zablotny, J. H. Xiao, I. Davidson, and J. C. Alwine, Mol. Cell. Biol. 13:961-969, 1993) suggested that T antigen could mediate transcriptional activation through interaction with the TATA-binding protein, as well as upstream bound transcription factors. Our present data are predicted by this model and suggest that at least one mechanism by which the T antigen manifests promiscuous transcriptional activation is its ability to interact with numerous transcription factors in a simple promoter context.