Pancreatic islets were prepared from control and vitamin D-deprived rats 2 or 5 weeks after weaning and, in the latter case, after 3 or 6 days treatment with exogenous vitamin D3 (60 nmol per day). The islets were prelabelled with both 86Rb and 45Ca and placed in a perfusion chamber. Vitamin D deprivation or administration failed to affect 86Rb outflow whether prior or after stimulation of the islets by a rise in either extracellular D-glucose or Ca2+ concentration. However, vitamin D deprivation decreased and vitamin D administration enhanced the basal 45Ca fractional outflow rate, as well as the magnitude of changes in both 45Ca and insulin release evoked by the rise in either D-glucose or extracellular Ca2+. It is proposed that the alteration in 45Ca fluxes and insulin release attributable to changes in the supply of vitamin D are, to a large extent, independent of the changes in nutrient catabolism conceivably associated with vitamin D deprivation and administration.