1 alpha,25-Dihydroxycholecalciferol [1,25-(OH)2D3] stimulated the proliferation and DNA synthesis of rabbit costal growth cartilage cells in the logarithmic growth phase in culture. The stimulatory effects of 1,25-(OH)2D3 were observable at a concentration of 10(-10) M and maximal at a concentration of 10(-8) M. On the other hand, 1,25-(OH)2D3 inhibited their expression of the cartilage phenotype, as judged morphologically, histochemically, and biochemically by a decrease in glycosaminoglycan (GAG) synthesis. The inhibition of GAG synthesis was also dose dependent and observable at a concentration of 10(-10) M. 1,25-(OH)2D3 also stimulated the proliferation of resting cartilage cells and inhibited their GAG synthesis, but its effects on these cells were less than those on growth cartilage cells, suggesting that 1,25-(OH)2D3 acts more specifically on growth cartilage cells than on resting cartilage cells. 1,25-(OH)2D3 had no effect on either DNA synthesis or GAG synthesis of growth cartilage cells in confluent cultures. 24R,25-Dihydroxycholecalciferol [24,25-(OH)2D3] had no effect on proliferation, DNA synthesis, or GAG synthesis of growth cartilage cells in the logarithmic growth phase. However, 24,25-(OH)2D3 had no effect on DNA synthesis of these cells in confluent cultures, but stimulated their expression of the cartilage phenotype. The stimulatory effect was dose dependent and maximal at 10(-7) M. Since chondrocytes express their differentiated phenotype as they become confluent in culture, these results suggest that 1,25-(OH)2D3 stimulates the growth of rapidly proliferating chondrocytes with a poorly differentiated phenotype and suppresses their expression of the cartilage phenotype, while 24,25-(OH)2D3 stimulates expression of the differentiated phenotype of highly differentiated chondrocytes.