AbstractSeveral proteins with important roles in oncogenesis have been shown to regulate the function of the E2F‐1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F‐1‐binding factor, from a yeast‐two hybrid screen using a portion of the E2F‐1 protein as “bait.” We demonstrate that the region within MDMX needed for the E2F‐1:MDMX interaction is located in the central part of the protein, C‐terminal of the p53‐binding domain. The region within E2F‐1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS‐PAGE, the faster migrating isoform having the stronger affinity for the E2F‐1 proteins. It appears that this interaction reduces the ability of E2F‐1 to bind DNA. Expression of MDMX along with E2F‐1 and Dp‐1 in Saos2 cells reduces the ability of E2F‐1 to bind to its consensus DNA sequence, without altering E2F‐1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F‐1 and negatively regulating its DNA binding ability. © 2002 Wiley‐Liss, Inc.