The simian virus 40 (SV40) transcriptional enhancer is composed of multiple cis-acting DNA sequence motifs, each individually having a two- to fourfold effect on the efficiency of transcription. When various distinct cis-elements act in combination, however, a dramatic enhancement of transcription initiation often results. SV40-enhancer A-domain sequences were previously shown to be important for early and late transcription in vivo. Here we report the isolation of the enhancer binding factor AP-4, which recognizes a motif in this domain. Purified AP-4 activates SV40 late transcription in vitro, and this stimulation is augmented by the addition of transcription factor AP-1 which binds to adjacent sequences in the A-domain, suggesting coordinate action of the two factors for transcriptional enhancement. AP-1 also represses late transcription from a major in vitro start site which is poorly used in vivo, indicating that AP-1 can act as both a positive and negative regulator of SV40 late transcription. Thus by manipulating the levels of different trans-acting factors in vitro, we can recreate the pattern of SV40 late initiation observed during the viral lytic cycle in vivo.