Puberty is associated with an increase in the plasma concentration of sex steroids, GH, and insulin-like growth factor-I (IGF-I). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induce skeletal growth is still not fully understood. To better understand the direct effect sex steroids have on bone growth, we studied an isolated organ culture system of the mandibular condyle, derived from 3.5-5.5-week-old male and female mice. We found that testosterone 10(-6) M, but not estradiol, stimulated thymidine incorporation into the DNA of male-derived condyle. Three days of testosterone treatment doubled the condyle size and increased the chondroprogenitor zone, while maintaining the normal gradient of the developing chondrocytes. Immunohistochemistry and in situ hybridization techniques showed that testosterone stimulated IGF-I and IGF-I-R and their messenger RNAs (mRNAs) mainly in the mature chondrocyte layer. Immunoneutralization of IGF-I in the testosterone-treated condyle caused the disappearance of the chondroblast and young chondrocyte layers, though the progenitor cell layer remained almost unaffected. Overtreatment with testosterone (dose or duration) accelerated condylar ossification. In the presence of testosterone 10(-5) M (high dose), calcification "climbs" up to the chondroprogenitor zone, and most of the condylar chondrocytes are replaced by bone tissue. Similar changes occurred after 7 days of testosterone treatment (long duration) with 10(-6) M. In conclusion, testosterone stimulates growth and local production of IGF-I and IGF-I-R in chondrocyte cell layers of an isolated organ culture of mice mandibular condyle. Part of the effect testosterone has on condylar growth is mediated by IGF-I.