Primary cultures of rat embryo fibroblasts have been shown to be resistant to transformation by dominant oncogenes such as v-src. We sought to determine if similar resistance is displayed by primary epithelial cells, and, if so, whether an immortalizing oncogene such as E1A could enhance transformation of primary epithelial cells by v-src. Transformation of primary rat epithelial cells by v-src was synergistically enhanced when E1A expression plasmids were cotransfected with a v-src expression plasmid. Foci were more numerous and observed earlier (9 to 14 days) with E1A plus v-src than with v-src alone (18 to 28 days). This cotransformation ability was abrogated by deletions in CR1 or CR2 of E1A, which encode the binding regions for the pRb family and are responsible for E1A-mediated cell cycle activation. Mutations in the p300 binding site or the second exon, which abolish immortalization, did not affect v-src cooperation, in contrast to ras and adenovirus E1B. While kinase activation was required for growth in soft agar, differential activation of Src kinase did not correlate with transformation efficiency. Cell morphology and actin structures were not dramatically impacted by E1A expression; thus, hypertransformation, as previously described for ras cotransformation, was not observed with v-src and second-exon mutants of E1A. However, growth rates for cells expressing both E1A and v-Src were higher than those for cells expressing only v-Src. These results suggest that functions involved in cell cycle activation encoded by E1A first exon can enhance v-src transformation of primary epithelial cells.