Postmenopausal hormone replacement therapy lowers colon cancer incidence. In humans, the mechanism is unknown, but animal models suggest that it may involve activation of the vitamin D receptor (VDR) pathway. The aims of our study were to determine whether estrogen intervention affects global gene expression in rectal mucosal biopsies and whether vitamin D-related genes are affected. Estradiol was given to raise serum estradiol to premenopausal levels in 10 postmenopausal women under close nutritional control. Primary end points were expression of VDR, CYP24A1, CYP27B1, and E-cadherin in rectal mucosa by reverse transcription-PCR and examining response to estradiol by genome-wide arrays. Responses in gene expression in rectal biopsies to estrogen were determined in each subject individually and compared with a human estrogen response gene array database and a custom array in vitro-generated database. Cluster analysis showed that subjects maintained their overall gene expression profile and that interindividual differences were greater than intraindividual differences after intervention. Eight of 10 subjects showed significant enrichment in estrogen-responsive genes. Gene array group analysis showed activation of the VDR pathway and down-regulation of inflammatory and immune signaling pathways. Reverse transcription-PCR analysis showed significant up-regulation of VDR and E-cadherin, a downstream target of vitamin D action. These data suggest that the chemopreventive action of hormone replacement therapy on colon neoplasia results, at least in part, from changes in vitamin D activity. Evaluation of gene arrays is useful in chemopreventive intervention studies in small groups of subjects.