The effects of T3 on cultured human fetal epiphyseal chondrocytes were assessed by studying its effects on DNA synthesis and alkaline phosphatase activity. DNA synthesis was evaluated as follows: after 48-h incubation in Ham’s F-12 serum-free medium, cultured chondrocytes were incubated with or without T3 (0.1-100 nM) in MCDB-104 serum-free medium for different periods of time (2-10 days), with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. Confluent cultured chondrocytes in 25-cm2 tissue culture flasks were incubated in Ham’s F-12 serum-free medium for up to 9 days with or without T3 (0.1-100 nM); the cellular cytoplasmic fraction was obtained, and alkaline phosphatase activity was evaluated using paranitrophenylphosphate as a substrate. No significant effects of T3 (0.1-100 nM) on DNA-[3H]thymidine incorporation were observed in any experiment (n = 17) for any gestational age (12-39 weeks) or for any incubation period studied (2-10 days). However, a significant (P less than 0.025 or more) stimulatory effect of T3 (0.1-100 nM) on alkaline phosphatase activity was observed after 9 days of incubation. This effect was highest for 5 nM T3 and was present in cultured chondrocytes from human fetuses of all ages studied (13-40 weeks). Cultured human fetal epiphyseal chondrocytes from human fetuses 12-40 weeks old (n = 8) showed specific nuclear binding sites for T3. The binding capacity was 27.14 ± 2.84 fmol/100 μg DNA, and the Kd was 0.66 ± 0.14 x 0.1 nM (mean ± SEM), with no significant differences among fetal ages. In conclusion, our results show that T3 elicits a biological response in cultured human fetal epiphyseal chondrocytes and has specific nuclear binding sites. Since alkaline phosphatase is closely related to the mineralization of epiphyseal cartilage, these results suggest that thyroid hormones could regulate this process. © 1992 by The Endocrine Society.