Adenovirus E1A proteins stimulate transcription by RNA polymerases II and III from many promoters. The detailed mechanism of transcriptional activation (transactivation) by E1A proteins remains unclear, but genetic and biochemical results suggest that E1A products might act to stimulate the activity of cellular transcription factors. In this study, a detailed mutational analysis of the adenovirus E1B promoter was undertaken to define the DNA sequences required for proper basal transcription and E1A transactivation. Two key findings emerged: first the E1B promoter is an unusually simple RNA polymerase II promoter requiring only two sequence elements for proper regulation, the TATA box and a binding site for transcription factor Sp1; and second only mutations in the TATA box interfere with E1A-transactivation, suggesting that E1A mediates its effect on this promoter through the TATA-box transcription factor.