1. Female rats fed on a vitamin D deplete (D-) diet for 90 days prior to mating gave birth to litters having significantly fewer young than females fed a diet containing vitamin D (2.2 IU/g, D + diet). 2. Neither age nor body weight of females had a significant influence on litter size. 3. Serum calcium and phosphorus levels in D+ and D- dams were not significantly different, nor were serum calcium and phosphorus levels and bone ash of D+ and D- weanlings. 4. The level of 25-hydroxyvitamin D3 [25(OH)D3], the main circulating form of vitamin D, was low in D- dams and was not detected in D- weanlings. 5. These data suggest that vitamin D is required for reproduction in the rat.