A model of low-phosphate, vitamin D-deficient rachitic rats was used to compare the effects of 1 alpha(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 on cartilage and bone. The rats were maintained for 3 weeks on a high-calcium, low-phosphate, vitamin D-deficient diet, during which period they developed severe rickets. The rachitic rats were injected for 2 or 3 consecutive days with a physiologic dose of either metabolite. Other littermates were given a single dose of 50,000 IU of cholecalciferol in combination with a normal diet. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscopic studies of the growth plate cartilage and bone. Treatment with 1 alpha (OH)D3 or with 1,25(OH)2D3, in spite of increasing Ca and P levels in the Cfl, induced only partial healing of the rickets. In contrast, 24,25(OH)2D3 or vitamin D with a normal diet resulted in complete morphologic and biochemical healing of the rickets. Transmission electron microscopic (TEM) studies have shown partial mineralization of the wide hypertrophic zone of the growth plate following treatment with 1 alpha(OH)D3 or with 1,25(OH)2D3. Mineralization was more complete with 24,25(OH)2D3 treatment. The results of this study emphasize the importance of 24,25(OH)2D3 for normal endochondral bone formation and mineralization.