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Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.