After weaning, rats were given free access to a control or vitamin D-deprived diet for 2 to 5 weeks. In the vitamin D deficient rats, plasma concentrations of 25-(OH)D3, 1,25-(OH)2D3,24,25-(OH)2D3 calcium, glucose and insulin were all decreased. After an overnight fast, the plasma insulin concentration was also decreased even when the plasma glucose concentration was not significantly affected. The food intake and body growth was also impaired in vitamin D-deficient rats. Administration of vitamin D3 in oil for 3 to 6 days to vitamin D-deficient rats increased the plasma concentration of vitamin D metabolites, calcium and insulin, but not that of glucose, and stimulated food intake and body growth to a larger extent than in rats treated by oil alone. Vitamin D deprivation decreased and vitamin D treatment increased the insulin content of the whole pancreas or isolated islets and the secretory response of the islets to D-glucose. The changes in insulin release remained significant when the hormonal output was expressed relative to the insulin content of the islets. These findings confirm that vitamin D deficiency causes alterations of pancreatic B-cell function. Moreover, the time course for changes in biological variables during vitamin D deprivation and treatment suggests that such an alteration cannot be solely accounted for by concomitant abnormalities in either plasma calcium or glucose concentrations.