A segment comprising the transcriptional enhancer elements was deleted from a recombinant plasmid carrying the simian virus 40 genome. The mutated viral chromosome was excised from the plasmid and propagated through several cycles of growth in monkey kidney cells. A variant was obtained that carried reiterations of sequences that span both sides of the deleted enhancer region. The mutant virus, dup 1495, displays a lag in its growth kinetics as compared to its parent, but it ultimately generates wild-type yields. The mutant virus expresses early mRNAs at near-normal levels, and the reiterated sequence functioned in cis to enhance transformation of mouse cells by the herpesvirus thymidine kinase gene. Thus dup 1495 reiterated segments encode enhancer activity even though their primary sequence is radically different from that of the normal simian virus 40 enhancer.