The observation that charcoal-treated fetal bovine serum (ctFBS) was able to modify one of main pathways of estrogens in cancer cells in culture, prompted us to initiate the present study. The active component of serum was isolated using native preparative polyacrylamide gel electrophoresis (PAGE). Under analysis with SDS-PAGE, a M(W) of 68 kDa and mobility of authentic bovine serum albumin (BSA) was observed. The addition of BSA to the serum free culture medium of HEC 1A human endometrial cancer cell line, resulted in an alteration of estradiol (E2) metabolism similar to that observed in the presence of ctFBS. BSA in fact, much enhanced 16 alpha-hydroxylation and significantly reduced 2-hydroxylation of E2 in HEC 1A cells. Comparable results were obtained with different endometrial (Ishikawa) and mammary (MCF-7) tumor cell lines having a different metabolic conversion rate of E2. Several albumin preparations from either bovine or human serum had the same effect; besides, BSA activity was unaffected by treatment with dextran-charcoal or heat. In the light of the present results, the inclusion of serum albumin (SA) in the formulation of media for studies evaluating steroid metabolism in cultured cells should be carefully considered.