Androgen receptors and 5 alpha-reductase activity were studied previously in genital skin fibroblasts cultured from normal subjects and patients with abnormalities of sex differentiation. We have now identified and characterized the androgen receptor in cultured human testis fibroblasts (HTF). HTF possess specific receptor proteins for androgens and translocate the receptor-steroid complex to nuclei. Approximately 50% of total cell binding was within nuclei, and 60-70% of nuclear binding was extracted by 0.5 M KCl (1 h; 0 C). Specific binding of dihydrotestosterone (DHT) was absent in HTF cultured from three patients with receptor-negative complete androgen insensitivity. 5 alpha-Reductase activity was very low (less than 100 pg 5 alpha-reduced products/micrograms DNA X h) in HTF after incubation with 200 nM [3H] testosterone (T). Based on this finding, androgen receptor binding of T was studied and resulted in a maximum binding capacity similar to that for DHT, but with a slightly lesser binding affinity (Kd). Binding to the receptor in HTF was specific for androgens (DHT, T, and R1881). [3H]DHT (2 nM) binding in the presence of 100 nM radioinert steroid was decreased by DHT (87%), R1881 (82%), and T (72%), but less with estradiol (53%), progesterone (31%), androstanediol (23%), and dexamethasone (10%). The androgen receptor in HTF was characterized as a macromolecule which sedimented at 4-5S on 0.4 M KCl sucrose density gradients and eluted as three high mol wt peaks on Sephacryl S-300 chromatography. Low but detectable aromatase activity was present in HTF and had the characteristics of being induced by glucocorticoid and having a Km similar to that of aromatase activity for genital skin fibroblasts. In summary, specific androgen receptors are present in HTF, and their characteristics are similar to those previously described for genital skin fibroblasts.