Adenovirus serotype 5 E1a proteins immortalize primary cells and in cooperation with products of a second oncogene, such as adenovirus serotype 5 E1b or EJ ras, produce full transformation. E1a also activates transcription of specific viral and cellular promoters, represses enhancer-dependent genes, and induces cellular DNA synthesis in quiescent cells. Comparison of different adenovirus serotypes has identified three conserved regions in the E1a protein sequence. We have analyzed E1a mutants with deletions-linker insertions in or preceding the first conserved region, region 1 (amino acids 40 through 77 of adenovirus serotype 5 E1a). E1a mutants which have in-frame deletions-substitutions in region 1 or pre-region 1 sequences were reconstructed into adenovirus to yield a total of 14 mutant viruses. All the mutant viruses showed wild-type growth in HeLa cells, confirming that region 1 is nonessential in these cells. However, we show that region 1 provides two distinct functions in infected primary rodent cells. One function is essential for induction of cell DNA synthesis, and the other is essential for focus formation. In addition, our results are consistent with a requirement for the DNA induction function in focus formation.