Androgens regulate the growth of many human hair follicles, but only pubic, axillary, and scalp hair growth occur in men with 5α-reductase deficiency. This suggests that 5α-dihydrotestosterone is the active intracellular androgen in androgen-dependent follicles, except in the axilla and pubis. Since the dermal papilla plays a major regulatory role in hair follicles and may be the site of androgen action, we have investigated androgen metabolism in six primary lines of cultured dermal papilla cells from pubic and axillary hair follicles; previous studies have shown that beard cells take up and metabolize testosterone, retaining and secreting 5α-dihydrotestosterone.After 24 h preincubation in serum-free Eagle's medium 199, 100-mm dishes of confluent cells were incubated for 2 h with 5 nM [1,2,6,7- 3 H]testosterone. Media were collected and the cells washed with phosphate-buffered saline and extracted with chloroform: methanol (2:1). After the addition of unlabeled and 1 4 C-labeled marker steroids, the extracts were analyzed by a two-step thin-layer chromatography system; steroid identity was confirmed by recrystallization to a constant 3 H 1 4 C ratio. Beard and pubic dermal papilla cells were also incubated for 24 h, and the medium was analyzed at various times.The results from pubic and axillary primary cell lines were similar. In both cells and media the major steroid identified was testosterone, but significant amounts of androstenedione were present, indicating 17β-hydroxysteroid dehydrogenase activity; androstanedione was also identified within the cells, but a small amount of 5α-dihydrotestosterone was only identified in one pubic cell line. Beard dermal papilla cells secreted large amounts of 5α-dihydrotestosterone into the medium over 24 h in contrast to pubic cells, which produced only very small amounts. The pubic and axillary cell results contrast with the observations of pronounced 5α-dihydrotestosterone in beard cells and confirm that androgen metabolism in cultured dermal papilla cells reflects the parent follicle's ability to respond to androgen in the absence of 5α-reductase type II in vivo. This supports our hypothesis that androgen acts on hair follicles via the dermal papilla and suggests that cultured dermal papilla cells may offer an important model system for studies of androgen action.