Cyclic ADP-ribose (cADPR) is a naturally occurring cyclic nucleotide and a potent mediator of calcium mobilization in many mammalian tissues. Previous studies have shown that cADPR is synthesized from beta-NAD(+) via the scission of the nicotinamide-ribose linkage and cyclization by forming a new bond between the ribose and the nitrogen of the adenine ring. However, the position and stereochemistry of this newly formed linkage were not unequivocally determined. In this study we have established that cADPR has the anomeric carbon of the ribose attached onto the N-1-nitrogen of the adenine nucleus via a beta-N-glycosyl linkage. The structural assignment was made by correlating cADPR to N-1-(5’-phosphoribosyl)AMP, a known intermediate of histidine biosynthesis. This was achieved by cleaving the pyrophosphate bond of cADPR under conditions (DMSO/tert-butoxide) not perturbing the integrity of the C-N glycosyl bond. Furthermore, cADPR was successfully synthesized by cyclization of N-1-(5’-phosphoribosyl)ATP catalyzed by NAD(+) pyrophosphorylase in an organic solvent-aqueous medium.