Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (SV40) core enhancer was ligated either adjacent to or 2.6 kb distant from the SV40 minimal origin of replication (ori) in both orientations. Plasmids were transfected into T antigen producing COS cells, and episomal DNA was harvested for quantitation of replication. Replication could be assessed either as accumulation of fmol of MboI sensitive progeny DNA, or as a transition In S DNA in replicated (MboI sensitive) versus unreplicated (Dpnl sensitive) form. The two measures were related exponentially (r=0.86). The SV40 enhancer augmented replication 1.5–10 fold. The effect was time dependent, distance dependent (only the adjacent enhancer locus stimulated replication), partially orientation dependent, and enhancer copy number independent. Phorbol ester did not affect replication. The heterologous glucocorticoid enhancer had no effect on replication. We conclude that the SV40 enhancer′s cis-effect on replication seems to be dependent on the close proximity to the replication origin of specific homologous sequences within the enhancer, rather than a typical enhancer-like effect.