The major immediate-early (MIE) transactivator proteins of cytomegaloviruses (CMV) play a pivotal role in the initiation of virus-host cell interactions. Therefore, cis- and trans-acting factors influencing the expression of these proteins through their upstream promoter-enhancer regions are important determinants of the outcome of virus infection. S1 nuclease analysis and in vitro transcription assays with the MIE (or IE94) transcription unit of simian CMV (SCMV) (Colburn) revealed a single prominent mRNA start site associated with a canonical TATATAA motif. This initiator region lies adjacent to a 2,400-bp 5'-upstream noncoding sequence that encompasses a newly identified 1,000-bp (A+T)-rich segment containing intrinsically bent DNA (domain C), together with the previously described proximal cyclic AMP response element locus (domain A) and a tandemly repeated nuclear factor I binding site cluster (domain B). Deleted MIE reporter gene constructions containing domain A sequences only yield up to 4-fold stronger basal expression in Vero cells than the intact simian virus 40 promoter-enhancer region, and sequences from position -405 to -69 (ENH-A1) added to a minimal heterologous promoter produced a 50-fold increase of basal expression in an enhancer assay. In contrast, neither the nuclear factor I cluster nor the bent DNA region possessed basal enhancer properties and neither significantly modulated the basal activity of the ENH-A1 segment. A second segment of domain A from position -580 to -450 was also found to possess basal enhancer activity in various cell types. This ENH-A2 region contains three copies of a repeated element that includes the 10-bp palindromic sequence CCATATATGG, which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response transcription factor. Reporter gene constructions containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells, but an ENH-A2 DNA segment encompassing two of the core serum response transcription factor binding sites failed to respond to serum induction in NIH 3T3 cells. Although there are overall similarities in the organizations of both the MIE enhancers and MIE transcription units among human CMV, SCMV, and murine CMV, the specific arrangements of repetitive motifs are quite different, and the bent DNA and ENH-A2 domains appear to be unique to SCMV.