The contribution of adenovirus early genes, other than that of the well-documented EIa immediate early gene, to the transcriptional regulation of the viral EIIa early transcription unit was examined. HeLa cells were transfected with EIIa-containing plasmids and co-transfected with distinct plasmids bearing one of the viral regions EIa, EIII or EIV. Co-transfection with the EIV-recombinants, but not the EIII constructs, stimulated specific transcription from the major EIIaE start site (EIIaE1) by 5- to 15-fold, as concluded from quantitative S1 nuclease analysis of cytoplasmic RNA and in vitro nuclear 'run-on' transcription assays. The extent of the EIV-induced stimulation was similar to that achieved by EIa under identical conditions. However, in contrast to our observations for EIa-mediated stimulation, where no unique EIIaE1 promoter elements were implicated, maximal induction by EIV requires sequences between positions -48 and -19 (with respect to the EIIaE1 start site).