Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the l-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the 13-globin gene. We observed a correlation between the number of inserted 26-mers and the level of 3-globin RNA expression.Transcriptional enhancers were originally identified within the genomes of DNA tumor viruses simian virus 40 (SV40) (2, 38) and polyomavirus (8, 9) as DNA segments that stimulate the transcription of linked genes in an orientationindependent manner and that function over long distances (16,26). Many other viral (19,20,22,[27][28][29][30][31][32] and several cellular (1, 14, 15, 41) enhancers have since been described. Although the general importance of enhancers in the regulation of transcription has been clearly demonstrated, numerous questions about their structure and function remain unanswered. There are striking DNA sequence homologies shared among several enhancers, but no unique consensus sequence common to all of them has emerged. Some enhancers contain direct tandem repeats of sequences about 70 base pairs (bp) in length. The repeats represent redundant copies of the same signal, because deletion of either does not alter transcriptional efficiency (2). Other enhancers lack direct repeats, but still involve elements dispersed over 100 to 300 bp that appear to be in some way functionally redundant. The polyomavirus enhancer was the first example of the latter situation (9, 46). This enhancer occurs within a 244-bp DNA fragment that also includes cis-acting elements essential for viral DNA replication. The sequences within the polyomavir...