The late promoter of simian virus 40 is transcriptionally activated, in trans, by large T antigen, the primary viral early gene product. Although large T antigen is a well-characterized DNA-binding protein, a variety of data suggest that its trans-activation function does not require direct interaction with DNA. We demonstrate that defined late promoter elements, omega (omega), tau (tau), and delta (delta), necessary for T-antigen-mediated trans-activation, are binding sites for simian cellular factors, not T antigen. Two of the late promoter elements (omega and tau) are shown to bind the same factor or family of factors. These factors bind to a site very similar to that for the HeLa cell factor AP1. We refer to these factors as the simian AP1-sequence recognition proteins (sAP1-SRPs). Compared with normal simian CV-1P cells, the sAP1-SRPs from T-antigen-producing COS cells, or from 14-h simian virus 40-infected CV-1P cells, showed altered binding patterns to both the omega and tau binding sites. In addition, the sAP1-SRPs from T-antigen-containing cells bound to the tau site more stably than did the analogous factors from normal CV-1P cells. The altered pattern of binding and the increased stability of binding correlated with the presence of T antigen in the cell. Additionally, the alteration of the binding pattern within 14 h of infection in CV-1P cells is temporally correct for late promoter activation. Overall, the data show (i) that the late promoter elements necessary for T-antigen-mediated trans-activation contain binding sites for simian cellular DNA-binding proteins; (ii) that the presence of T antigen causes alterations in the binding characteristics of specific simian cellular DNA-binding factors or families of factors; and (iii) that factors which bind to the late promoter elements required for activation have altered and more stable binding characteristics in the presence of T antigen. These points strongly suggest that T antigen mediates trans-activation indirectly through the alteration of binding of at least one specific simian cellular factor, sAP1-SRP, or through the induction of a family of sAP1-SRP factors.