Adenovirus E1A genes possess transcriptional activation and repression activities. Three major gene products have been characterized, derived from the 13S, 12S and 9S mRNAs. Using transient expression assays in HeLa cells, we have investigated the effect of these gene products on the activity of the nine major Ad2 (or Ad5) promoters driving the expression of a reporter gene. Based on the results, we could separate the promoters into three classes: (a) E1A, which is active by itself, and is unaffected or slightly stimulated by the E1A 13S product (depending on the HeLa cell line used); (b) the other classical early promoters (E1B, E2e, E3, E4), all of which are active alone and are stimulated by the 13S product and repressed by the 12S product; and (c) the late promoters (IX, IVa2, MLP, E2L) which are not active alone and are substantially unaffected by the 13S or 12S products. Thus the 13S and 12S gene products have antagonistic effects on at least four adenovirus promoters. The 9S product did not influence the activity of any of the adenovirus promoters. Upon transfection into 293 cells, all the early promoters were active and all the late promoters were inactive, except for the major late promoter (MLP). We demonstrate that the combination of the E1A and E1B genes is a potent activator of the MLP in HeLa cells and discuss these results in the context of the infectious cycle.