Inositol 1,4,5-trisphosphate (IP3) is thought to be a second messenger for intracellular calcium mobilization. However, in a cell-free system of islet microsomes, cyclic adenosine diphosphate-ribose (cADP-ribose), a nicotinamide adenine dinucleotide (NAD+) metabolite, but not IP3, induced calcium release. In digitonin-permeabilized islets, cADP-ribose and calcium, but not IP3, induced insulin secretion. Islet microsomes released calcium when combined with the extract from intact islets that had been incubated with high concentrations of glucose. Sequential additions of cADP-ribose inhibited the calcium release response to extracts from islets treated with high concentrations of glucose. Conversely, repeated additions of the islet extract inhibited the calcium release response to a subsequent addition of cADP-ribose. These results suggest that cADP-ribose is a mediator of calcium release from islet microsomes and may be generated in islets by glucose stimulation, serving as a second messenger for calcium mobilization in the endoplasmic reticulum.