To investigate the effect of oral and transdermal estrogen replacement therapy (ERT) on the expression of different estrogen receptor (ER) subtypes and the secretion of immunoregulatory cytokines, we performed a clinical investigation on previously healthy women who had undergone a total hysterectomy and bilateral salpingo-oopherectomy. These women were randomly distributed into two groups: an oral ERT group and transdermal ERT group. Before and after ERT, the serum levels of estradiol (E 2 ) and follicle stimulating hormone (FSH) were measured, ERα and ERβ expression of peripheral blood T lymphocytes was tested, and secretion of specific immunoregulatory cytokines (IFNγ, IL-2 and IL-4) by T lymphocytes was examined. Our results confirm that for both groups, the serum E 2 level was increased after ERT (P<0.01) and the serum FSH level was decreased after ERT (P<0.01), with no significant difference in hormone levels between the two groups. ERα expression by T lymphocytes was significantly higher after ERT than before (P<0.01) in both groups. Levels of type 1 cytokines (IL-2 and IFNγ), which were secreted by T helper 1 (Th1), after ERT were substantially decreased. The level of type 2 cytokine (IL-4), which were secreted by T helper 2 (Th2), was significantly increased after ERT (P<0.01 for the oral group and P<0.05 for the transdermal group). In summary, both oral and transdermal ERT increased serum E 2 levels, decreased serum FSH levels and relieved the effects of peri-menopausal symptoms. These data suggest that both oral and transdermal ERT act to improve the balance of Th1/Th2 cytokines by the effects of estrogen potentially acting in T lymphocytes mainly through ERα.