Cyclic adenosine diphosphoribose (cADPR), a recently discovered metabolite of nicotinamide adenine dinucleotide (NAD), is a potent calcium-releasing agent postulated to be a new second messenger. An enzyme that catalyzes the synthesis of cADPR from NAD and the hydrolysis of cADPR to ADP-ribose (ADPR) was purified to homogeneity from canine spleen microsomes. The net conversion of NAD to ADPR categorizes this enzyme as an NAD glycohydrolase. NAD glycohydrolases are ubiquitous membrane-bound enzymes that have been known for many years but whose function has not been identified. The results presented here suggest that these enzymes may function in the regulation of calcium homeostasis by the ability to synthesize and degrade cADPR.