The phenomenon of chondrocyte hypertrophy is accompanied by the expression of type X collagen and the appearance of matrix mineralization. These events are also associated with changes in the phosphorylation of intracellular proteins. In this study the addition of 10 mM beta-glycerophosphate to hypertrophic chondrocytes resulted in stimulation of type X collagen synthesis up to 10 days in culture and an increase in the expression of type X collagen mRNA. This was followed by the onset of mineralization and the appearance of calcium hydroxyapatite. In contrast, the addition of beta-glycerophosphate to non-hypertrophic chondrocytes failed to induce expression of type X collagen or to produce changes in calcium and phosphate. The increased formation of type X collagen and of mineral in hypertrophic chondrocytes was accompanied by changes in the tyrosine kinase pp60c-src. While the level of c-src protein decreased approximately 2.5-fold in hypertrophic chondrocytes after 17 days of beta-glycerophosphate treatment, the specific activity of pp60c-src kinase increased approximately 3-fold in the cells that could be induced to mineralize but remained unchanged in cells that did not exhibit this property. Regulation of kinase activity may be an important event in endochondral ossification.