Plasma levels of the vitamin D metabolites were related to changes in bone morphology during the development of rickets in rats deprived of phosphorus and vitamin D. Weanling rats were studied at 1, 3, and 5 wk after onset of diets deficient in phosphorus or in both phosphorus and vitamin D. Bone histology and morphometry were carried out and measurements were made of 45Ca and 32P absorption, serum Ca and P, and plasma 25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3. After 1 wk of vitamin D restriction, the plasma levels of 25(OH)D3 and 24,25(OH)2D3 were non-detectable (less than 0.5 and less than 0.8 ng/ml). The plasma 1,25(OH)2D3 level was elevated at 1 wk (105.5 pg/ml) and fell to 19 pg/ml by 5 wk. At 1 wk mild rachitic lesions in epiphyseal cartilage were observed despite the elevated 1,25(Oh)D3 level. Serum Ca and P levels and values for 45CA and 32P absorption decreased and the severity of the rickets increased with the fall in plasma levels of 1,25(OH)2D3. In Vitamin D replete, phosphate deficient rats the epiphyseal cartilage was normal throughout the 5 wk study period. Our results provide further evidence that physiological levels of 1,25 (OH)2 D3 will not prevent rickets without adequate plasma concentrations of either 25(OH)D3 or 24,25(OH)2D3.