Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.