The adenovirus-2 E1A 289R transcription activator protein contains a 49 amino acid sequence (designated CR3) that has been suggested to represent the minimal domain required for E1A-induced activation of viral early transcription. We show here that the non-conserved carboxy-terminal E1A exon contains two interchangeable elements that are required for efficient CR3-dependent transactivation of the adenovirus E4 promoter in HeLa cells. These two elements do not encode independent transactivation functions and have been designated auxiliary regions (ARs) 1 and 2. The effects of AR1 and AR2 are not additive, suggesting that they contribute a mechanistically analogous function in transcription. Previous studies have suggested that two cellular transcription factors, ATF-2 and E4F, can function together with E1A to induce transcription of the E4 promoter. The importance of respective factors for E4 transcription has not been resolved. We find that E1A activation of E4F, but not ATF-2 (or other ATF factors), is AR1- and AR2-dependent. This result suggests that E1A induction of the E4 promoter in HeLa cells is primarily mediated by E4F.