Targeted deletion of genes encoding the 1,25-dihydroxyVitamin D [1,25(OH)(2)D]-synthesizing enzyme, 25 hydroxyVitamin D-1alpha-hydroxylase [1alpha(OH)ase or CYP27B1], and of the nuclear receptor for 1,25(OH)(2)D, the Vitamin D receptor (VDR), have provided useful mouse models of the inherited human diseases, Vitamin D-dependent rickets types I and II. We employed these models and double null mutants to examine the effects of calcium and of the 1,25(OH)(2)D/VDR system on skeletal and calcium homeostasis. Optimal dietary calcium absorption required both 1,25(OH)(2)D and the VDR. Skeletal mineralization was dependent on adequate ambient calcium but did not directly require the 1,25(OH)(2)D/VDR system. Parathyroid hormone (PTH) secretion was also modulated primarily by ambient serum calcium but the enlarged parathyroid glands which the mutants exhibited and the widened cartilaginous growth plates could only be normalized by the combination of calcium and 1,25(OH)(2)D, apparently independently of the VDR. Optimal osteoclastic bone resorption and osteoblastic bone formation both required an intact 1,25(OH)(2)D/VDR apparatus. The results indicate that calcium cannot entirely substitute for Vitamin D in skeletal and mineral homeostasis but that the two agents have discrete and overlapping functions.