Vitamin D and phosphate deficiency has been shown previously to result in an accumulation of calcium acidic phospholipid phosphate complexes in the epiphysis of immature rats and to increase the cholesterol and decrease the lysophosphatide content throughout bone. To define the effects of vitamin D deficiency and those of combined vitamin D and phosphate deficiency, the lipid composition of the bones of second generation rachitic rats, raised on a diet that was vitamin D deficient was compared to that of normal animals fed a vitamin D-repleted diet and to previously studied vitamin D and phosphate-deficient rats. Animals raised on the vitamin D-deficient diet in utero and during postfetal life were serologically quite different from controls. These animals were significantly hypocalcemic and had no detectable vitamin D metabolites. Similar to the previously studied phosphate-deficient rachitic animals, the vitamin D-deficient animals had significantly elevated total lipid contents (weight percent lipid per demineralized dry weight) throughout their bones as compared to controls (epimetaphysis 9.6 vs 6.7 (P less than or equal to 0.03), diaphysis 6 vs 2 (P less than or equal to 0.01), calvaria 6.1 vs 2.4 (P less than or equal to 0.002). Similar to the vitamin D and phosphate-deficient animals, the second generation vitamin D-deficient rat bones had elevated cholesterol and reduced lysophosphatide contents. Complexed acidic phospholipids were not significantly elevated in the vitamin D-deficient animal bones. The data suggest that the lipid changes seen in these animals are due to vitamin D deficiency and not to phosphate deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)