To establish the role of vitamin D in bone metabolism during the mammalian reproductive cycle, female rats were maintained on a vitamin D-deficient diet from weaning and mated with normal males. Changes in the femurs of the females during pregnancy and lactation and after weaning were monitored. The concentration of calcium in the plasma of mature vitamin D-deficient rats was 5.5-5.8 mg/100 ml. This was reduced to 3.8 and 4.4 mg/100 ml by day 14 of lactation and weaning, respectively. Total femur calcium and calcium per unit volume were approximately two thirds of that observed in agematched vitamin D-replete rats. During pregnancy, little or no change was observed in femurs from vitamin D-replete females. Bone volume, total bone calcium, and bone calcium per unit volume in vitamin D-deficient females, however, tended to increase during pregnancy. After parturition, both vitamin Dreplete and -deficient females lost bone mineral. When based on an initial value equivalent to that on day 20 of pregnancy, the losses were similar and amounted to 42–46 mg calcium or 23– 38% of the total bone mineral. After weaning, there was a trend toward replacement of bone mineral in vitamin D-replete females. These results indicate that vitamin D is not required for bone calcium mobilization during lactation and suggest that other as yet unidentified factors may play a key role in bone metabolism during reproduction.