The metabolic activity of skin is important in penetration of topically applied compounds. Currently, animal or cadaver skin is used to evaluate the relationship between metabolism and penetration. In the present study, testosterone metabolism and penetration in a three-dimensional human skin model consisting of keratinocytes and fibroblasts derived from neonatal foreskins was characterized. Pieces of the model were placed on tissue culture inserts with HEPES-buffered medium on the dermal side. Penetration of [3H]testosterone was faster at 32 degrees C than 4 degrees C suggesting that metabolism affected penetration. To evaluate this metabolism, [3H]testosterone was applied to the stratum corneum side of the skin model. Radiolabeled metabolites released into the medium after incubation were separated by HPTLC and analyzed by autoradiography. This skin model metabolized [3H]testosterone to both more polar and non-polar compounds which were similar to metabolites of neonatal foreskins. The appearance of non-polar compounds was earlier than the appearance of polar compounds. Both dermal fibroblasts and differentiated epidermal keratinocytes contributed to the metabolism of testosterone. Two testosterone metabolites, dihydrotestosterone and androstane-3, 17 diol, were reduced by addition of the cytochrome P-450 inhibitor metyrapone and were only produced by the keratinocytes. In conclusion, this model is a reproducible source of metabolically active skin and therefore a good alternative to animal or cadaver skin for evaluation of the contribution of metabolism to penetration