Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E₂) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E₂ and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [³H] labeled E₂ was converted to E₂ sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E₂ at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E₂ inactivation is primarily mediated by SULT1E1 in keratinocyte and E₂ action is likely suppressed in epidermal differentiation.