We describe here changes in the regulatory region of SV40 that influence its growth potential in cultured cells. Laboratory strains of papovaviruses BK and JC differ in the sequence of their regulatory regions from archetypes that have not been passaged in cell culture. These archetypes lack sequence repeats in the regulatory region; duplications that occur upon passage in cell culture confer selective growth advantage. Changes within the enhancer-promoter region of the well-characterized 776 strain of papovavirus SV40 that might affect its growth in tissue culture cells have not been documented. We measured the effect upon the growth of SV40 (776 strain) in CV-1 cells either of adding an additional 72-basepair (bp) enhancer element or of duplicating the entire 21-bp repeat region. SV40 growth in tissue culture was improved by reiteration of enhancer elements, whereas no growth advantage was conferred by tandem duplication of the 21-bp repeats. Viral DNA infectivity in CV-1 cells was directly proportional to the number of 72-bp elements but was unaffected by tandemly repeated 21-bp repeat elements. This study suggests that the 776 strain of SV40 is an evolutionary intermediate and that tissue-culture-adapted strains of SV40 do not accurately reflect the replication potential of natural isolates from primate hosts.