The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occured by any of the following events: (i) restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 ElA enhancer core AGGAAGTGACT; (ii) acquisition of an A--G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; (iii) duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with inmunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein.Transcriptional enhancers are cis-acting DNA elements that stimulate gene expression. The distinguishing feature of enhancers is their ability to activate RNA polymerase II transcription of linked genes in a relatively orientation-and distance-independent fashion (2, 3, 49). Transcriptional enhancers were first detected in simian virus 40 (SV40) and polyomavirus (2,3,15,28,49), and they have since been found in the genomes of numerous other DNA and RNA tumor viruses, as well as in cellular genes (reviewed in references 57 and 65). Some yeast genes have upstream activator sequences whose function resembles that of enhancers (29). Enhancer activity is often restricted to particular species and tissues (14,24,34,41,42,64). This ubiquity and specificity of action indicates that enhancers play a central role in the control of eucaryotic gene expression. However, the mechanism of enhancer action is unknown.The polyomavirus enhancer region is required in cis both for early gene expression (23,41,50,73,75) and for DNA replication (16,24,46,51,75). Deletion analyses indicate that the enhancer contains sequences which are functionally redundant in fibroblasts (30,73,75 evidence suggest that the replicative and transcriptional functions of the enhancer are linked.Other functionally important segments of the polyomavirus enhancer lie within the PvuII-PvuII fragment (nt 5152 to 5289, termed PvuII-4) adjoining the origin of DNA replication. Short sequence motifs within this fragment are also present in the immunoglobulin G (I...