The conserved region 3 (CR3) portion of the human adenovirus (HAdV) 5 E1A protein functions as a potent transcriptional activator that induces expression of viral early genes during infection. Expression of HAdV-5 CR3 in the yeast Saccharomyces cerevisiae inhibits growth, as do the corresponding regions of the HAdV-3, 4, 9, 12 and 40 E1A proteins, which represent the remaining five HAdV subgroups. Growth inhibition is alleviated by disruption of the SAGA transcriptional regulatory complex, suggesting that CR3 targets the yeast SAGA complex. In yeast, transcriptional activation by several, but not all, of the CR3 regions requires the Gcn5 acetyltransferase component of SAGA. The CR3 regions of HAdV-3, 5, 9 and 40, but not HAdV-4 and 12 interact with the pCAF acetyltransferase, a mammalian ortholog of yeast Gcn5. Disruption of the previously described N-terminal pCAF binding site abrogates binding by the HAdV-5 243R E1A protein, but not the larger 289R E1A protein, which is otherwise identical except for the presence of CR3. RNA interference directed against pCAF decreased HAdV-5 CR3 dependent transcriptional activation in mammalian cells. Our results identify a second independent binding site for pCAF in E1A and suggest that it contributes to CR3 dependent transcriptional activation.