When growth cartilage from rachitic chicks was cultured in the presence of the calcium-regulating hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), collagen resorption was increased and collagen synthesis decreased compared to control cultures containing no hormone. The minimum concentration of the hormone that caused a statistically significant inhibition of collagen synthesis was 10(-8) mol/l. Collagen synthesis by growth cartilage from normal chicks was also reduced by 1,25-(OH)2D3, showing that it was not an abnormal response of vitamin D-depleted tissue. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 also inhibited collagen synthesis by cultures of growth cartilage but only at higher metabolite concentrations. 1,25-Dihydroxyvitamin D3 (10(-7) mol/l) did not significantly inhibit collagen synthesis by cultures of articular fibrocartilage and of sternal cartilage, tissues that do not calcify physiologically. The minimum concentration of 1,25-(OH)2D3 (10(-9) mol/l) necessary to cause decreased collagen synthesis by embryonic chick calvaria was lower than the value obtained with growth cartilage; this suggests that bone cells may be more sensitive to the hormone in this respect than are growth cartilage chondrocytes. These findings provide evidence of a direct role of 1,25-(OH)2D3 in the control of endochondral bone formation which is consistent with its primary role in the maintenance of plasma calcium homeostasis.