The biological effects of dihydrotestosterone (DHT) and testosterone (T) on cultured human fetal epiphyseal chondrocytes were assessed by studying the ability of these androgens to promote DNA synthesis. DNA synthesis was evaluated by measuring [3H]thymidine incorporation into DNA. After 48-h incubation in Ham’s F-12 serum-free medium, chondrocytes were incubated with or without DHT (10-11-10-8m) or T(10-11-10-8m) in MCDB-104 serum-free medium for a further 48 h, with the addition of [3H]thymidine (5 μCi/mL) for the last 24 h. In chondrocytes from five male fetuses (12–40 weeks’ gestation) DHT and T significantly stimulated DNA synthesis. The maximum stimulatory effect was obtained for DHT at 10-10m (P < 0.01) and for T at 10-9m (P < 0.02). In chondrocytes from four female fetuses the stimulatory effect was significant only for DHT and was maximum at 10-10m (P < 0.02), whereas no effect was observed for T. Cultured chondrocytes from both male and female fetuses show the presence of proteins with high affinity and limited binding capacity (Bmax) for DHT (male fetuses: Bmax, 4.9 ± 1.9 × 10-15m/mg protein; Kd, 0.43 ± 0.24 × 10-9m; female fetuses: Bmax) 4.8 ± 1.6 × 10-16m/mg protein; Kd, 0.63 ± 0.19 × 10-9m) with no significant differences between sexes. In conclusion, our results show that androgens elicit a biological response in cultured human fetal epiphyseal chondrocytes and that DHT-binding sites are present in these cells. DHT, rather than T, seems to be the active androgen. A sex difference in the degree of androgen action is also documented. © 1990 by The Endocrine Society.