Two distinct nonoverlapping enhancer elements can be defined within the polyoma enhancer region. In mouse fibroblasts, element A provides a 3-fold higher enhancement of the alpha 2-collagen promoter than element B. In mouse embryonal carcinoma cells, element B shows the same efficiency as in fibroblasts, whereas that of element A decreases by a factor of 3.5. Moreover, a single point mutation (PyEC F9.1) increases the efficiency of element B in both cell types, making it superior to element A in embryonal carcinoma cells. The core of element A is located within a 35 bp region tandem duplicated in several wild-type strains, mostly homologous to a crucial repeated sequence of adenovirus E1a enhancer (Hearing and Shenk, 1983). Element B contains the consensus sequence of Weiher et al. (1982). These two homologies are precisely adjacent to the two DNAase l-hypersensitive sites present in viral chromatin.