Overexpression of the oncoprotein MDM2, a negative feedback regulator of p53, is often observed in breast cancer tissue and cell lines, particularly in those which express estrogen receptor alpha (ER alpha). In this study, we report a novel function of MDM2, i.e., as a positive regulator of ER alpha. This function does not involve p53. MDM2 overexpressing clones derived from the breast cancer cell line, MCF-7 cells, showed a remarkable growth advantage only in estradiol supplemented conditions, and this profile coincided with increased transcriptional activity of ER alpha in these cells. Though p53 has been reported to be an inhibitor of ER alpha function, p53 protein in MDM2 overexpressing clones was more abundant than in the parental cells. When ER alpha was exogenously expressed in p53-null cells, its activity was enhanced by coexpression of MDM2. Mammalian two-hybrid assays and GST pull-down assays indicated that MDM2 could interact with ER alpha. These results indicate that MDM2 is a direct activator of ER alpha function, and suggest such a role for MDM2 in ER alpha -positive breast cancer.