Adenovirus early region 1A(E1A) encodes a heterogeneous family of proteins some of which function as transactivators and are required for efficient viral replication in HeLa cells. We have constructed adenovirus type 5 (Ad 5) mutants in which the E1A transcription unit is placed under the control of either the human beta-actin promoter or the human cytomegalovirus immediate early promoter. The level of E1A expression in cells infected with these mutants was several times higher than that in wild-type virus infections. When the same heterologous promoters were inserted upstream of, but in the opposite orientation to, the E1A transcription unit, the level of expression was greatly reduced with respect to wild-type levels of E1A. Despite this variation of at least 40-fold in the concentration of E1A proteins in infected cells, there was no significant difference between wild-type Ad 5 and any of the mutants in their ability to replicate in HeLa cells. These results suggest that very low levels of E1A proteins are sufficient for virus production in cultured cells and that wild-type Ad 5 produces an amount of E1A in excess of that required.