In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5 alpha-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10(-6) M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10(-10) and 10(-9) M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 +/- 1.2 pmol/mg protein/day (mean +/- SEM), while the maximal stimulation 1043 +/- 46 pmol/mg protein/day was obtained at the concentration of 10(-8) M DHT. This stimulation was partially blocked with cyproterone acetate at level of 20 +/- 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.