We have assayed the cell-specific activity of a matched set of four enhancers found in viral revertants derived from simian virus 40 (SV40) enhancer mutants. These enhancers all contain 71-base-pair duplications that span identical regions or, in one case, the same region shifted by 2 nucleotides. The four enhancers differ, however, in that each one either carries a different wild-type pair of the genetically defined SV40 enhancer A, B, or C elements, with the other two elements mutated, or carries all three elements mutated. The three enhancers carrying two copies of a wild-type element effectively enhance transcription in CV-1 and HeLa cells, but only the enhancer containing a duplicated wild-type C element exhibits activity in NIH 3T3 cells. These results show that the ability of the A, B, and C elements to compensate for one another is cell specific and that selection for enhancer function in one cell type can generate enhancers with different cell-specific activities. These results are consistent with the hypothesis that tandem duplication of multiple distinct enhancer elements, as in wild-type strains of SV40 (e.g., the 72-base-pair repeat), has the property of expanding the host range of an enhancer.