Our purpose was to assess 17beta-hydroxysteroid dehydrogenase (17HSD) type 1 protein expression in normal breast tissue during the menstrual cycle and hormonal contraception. We analyzed 17HSD type 1 protein expression by immunohistochemistry during the regular menstrual cycle (n = 12) and hormonal contraception (n = 7) in women undergoing reduction mammoplasty. 17HSD type 1 protein was detected in normal breast epithelial cells throughout the menstrual cycle and in all women using hormonal contraception. Mean 17HSD type 1 staining intensity was higher in alveolar epithelial cells in women using hormonal contraception (2.14) than in untreated women (1.25; P < 0.04). For ducts, this difference approached significance (2.29 vs. 1.41; P = 0.06). There was a negative correlation between serum estradiol (E2) levels and 17HSD type 1 protein expression for both alveolar (r(s) = -0.68; P = 0.004) and ductal (r(s) = -0.75; P = 0.002) breast epithelial cells. Enhanced 17HSD type 1 protein expression might increase the conversion to E2 in normal breast tissue during hormonal contraception. The negative correlation between serum E2 levels and 17HSD type 1 suggests this enzyme to be one of the regulatory mechanisms of intratissue E2 concentration in normal breast tissue.