The tax protein of HTLV-II increases the level of steady-state mRNA produced from the HTLV-II long terminal repeat (LTR) and also activates heterologous promoters. We have previously shown that the adenovirus E3 promoter, which is trans-activated by the adenovirus E1a protein, is also trans-activated by the tax protein. To investigate the mechanism of trans-activation by tax, we analyzed E3 promoter deletion mutants to determine nucleotide sequence requirements for activation of this promoter. Our results show that removal of different upstream regions within the promoter does not result in loss of trans-activation, indicating that tax does not appear to interact with a single DNA binding protein to activate the E3 promoter. In addition, tax and E1a together activate the E3 promoter in a greater than additive fashion, suggesting that these proteins function differently. Possible mechanisms of activation by the tax protein are discussed.