The Living Skin Equivalent (LSETM) is an organotypic coculture of human dermal fibroblasts in a collagen-containing matrix and a stratified epidermis composed of human epidermal keratinocytes. In order to establish the feasibility of using this in vitro system as a model for cutaneous biotransformation, the metabolic fate of topically applied testosterone (T) was monitored in the LSE. After a 24-hour exposure period (37 °C) to radiolabelled T, LSE extracts analyzed by high-performance thin-layer chromatography showed that approximately 50% of the applied T had been metabolized. Identified metabolites included bands which comigrated with polar metabolites and products of T 5α-reductase. The general distribution of the observed metabolites was similar to that obtained usingbiopsied human skin. The rates of T penetration (32 °C) through the LSE were monitored after application of T in two vehicles (water and petrolatum) and yielded permeability constants (Kps) of 29 and 1.6 × 10-3 cm/h, respectively. These Kp values were 4- to 6-fold higher than those reported for human abdominal skin, and reflect the vehicle-related shift in penetration seen in human skin. The Kp values for two additional steroids, estradiol and hydrocortisone, and for T were also determined at 22 °C and compared to published Kp values. These Kp values in the LSE were, respectively, 63-, 187-and 35-fold higher than those reported for human skin. The data suggest that compared to human skin the LSE has only a partial barrier function to the passage of test chemicals. The magnitude of the difference in the rates of penetration between the LSE and human skin depends on the test chemical. For specific chemicals, such as T, the LSE simulates certain aspects of skin metabolism and percutaneous absorption.