The enhancer of simian virus 40 (SV40) is active in a wide variety of tissues and hosts and thus has been called a general enhancer. Previous work has established that this region contains a number of subsegments, each with a different cell type specificity. By analyzing enhancer deletion mutants of SV40 with a restricted cell type specificity, we have identified a purine-rich DNA motif (Pu box) that is essential for activity of the mutant enhancer in lymphoid cells and also obtained evidence for an independent element that confers activity in kidney CV-1 cells. The Pu box element is positively regulated since binding of a cognate factor(s) was only observed in lymphoid cell extracts. The Pu box is also present in the 63-bp enhancer repeat of lymphotropic papovavirus (LPV), and competition experiments show that the SV40 and LPV sequences bind the same factor(s). Thus, the Pu box is likely to be a major determinant of the lymphotropic host range of LPV. In SV40, however, the Pu box is one of several enhancer elements with different cell specificities. We suggest that such an arrangement reflects a diversification strategy of SV40 to maximize its chance of adaptation to various types of host cells.