The simian DNA tumor virus SV40 regulates synthesis of early and late viral transcripts from two divergent and overlapping promoters. The SV40 early promoter exhibits all the hallmarks of eukaryotic promoters, containing elements that function when positioned near the start site of transcription and other positionally flexible elements that constitute a transcriptional enhancer. The architecture of the SV40 enhancer has been elucidated by four complementary approaches: (1) saturation mutagenesis in vitro; (2) viral genetic selection in vivo; (3) synthetic multimerization of its component parts; and (4) detailed protein binding studies. The SV40 enhancer represents a cluster of cell-specific regulatory elements that cooperate with one another in a functional hierarchy. The basic units of enhancer structure, enhansons, are the building blocks and represent protein binding sites. The functional units are proto-enhancers, which consist of one or more enhansons. Proto-enhancers serve as regulatory modules that can cooperate with one another or duplicates of themselves in flexible arrangements to create a potent enhancer.