Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.