Steroid 5α-reductase is of crucial importance in androgen physiology because it catalyzes the conversion of testosterone into the more potent 5α-dihydrotestosterone in androgen-regulated target tissues. The enzyme occurs in two isoforms, whereby type 1 isozyme exists mainly in the skin and type 2 in the prostate. By using human cell cultures, we examined cutaneous expression and subcellular localization of type 1 5α-reductase in vitro. In immunocytochemistry, type 1 5α-reductase was detected in the cytoplasm of cultured human sebocytes, keratinocytes, fibroblasts, dermal microvascular endothelial cells, hair dermal papilla cells, and melanocytes. In western blot studies, two closely lying bands of 21–27 kDa were detected, possibly indicating heterogeneity of the type 1 5α-reductase in all the cell types tested, with the exception of beard dermal papilla cells. Northern blot studies revealed most abundant type 1 mRNA in neonatal foreskin keratinocytes, followed by adult facial sebocytes. Occipital hair dermal papilla cells presented higher levels of type 1 5α-reductase mRNA than those of beard. These findings were confirmed by semiquantitative reverse transcriptase polymerase chain reaction coupled with high performance liquid chromatography analysis. Taken together, it seems likely that in cultured human skin cells there exist (i) heterogeneity of type 1 5α-reductase protein and (ii) quantitative differences in its transcriptional and translational expression levels.