Although adenovirus early region 1A (AdE1A) can modulate protein expression through its interaction with transcriptional regulators it can also influence the ability of the cell to degrade proteins by binding to components of the 26 S proteasome. We demonstrate here that AdE1A interacts with the S2 subunit of the 19 S regulatory complex in addition to the ATPase subunits S4 and S8 previously identified. S2 forms complexes with both the 13 and 12 S AdE1A proteins both in vivo and in vitro. Mutational analysis has shown direct binding through a short sequence toward the N terminus of conserved region 2 of AdE1A, which encompasses the LXCXE motif, involved in interaction with the pRb family of proteins. In vivo, additional contacts are made between AdE1A and proteasomal components, as well as within the proteasome, such that deletion of the N-terminal region of E1A as well as part of conserved region 2 is required to completely disrupt S2 binding. Mutation of AdE1A, which disrupts complex formation with S2, results in the loss of its ability to stabilize the p53 protein. Similarly down-regulation of S2 expression using small interfering RNAs leads to the inhibition of p53 degradation. These effects were observed in normally growing cells and those subjected to UV irradiation. Furthermore, AdE1A had no effect on the Mdm2-mediated ubiquitination of p53. We suggest therefore that interaction of AdE1A with S2, as well as with the ATPases S4 and S8, directly causes inhibition of proteasomal activity and consequent increase in the protein levels of p53.