We have synthesized 8-azido-cyclic ADP-ribose (8N3-cADPR) and [32P]8-azido-cyclic ADP-ribose ([32P]8N3-cADPR) in order to characterize cyclic ADP-ribose-(cADPR) binding sites in sea urchin egg homogenates. 8N3-cADPR was an antagonist of cADPR since it did not induce Ca2+ release from egg microsomes but did inhibit the ability of cADPR to do so. The effect of 8N3-cADPR was reversible and could be overcome by high concentrations of cADPR, suggesting that both were acting on the same site. This was supported by the fact that 8N3-cADPR effectively competed for [32P]cADPR binding to microsomes. Reciprocally, binding of [32P]8N3-cADPR could also be selectively displaced by cADPR and 8N3-cADPR, but not by ADP-ribose. These results indicate that 8N3-cADPR binds specifically to the cADPR-binding sites and inhibits cADPR from releasing Ca2+. Photolysis of microsomes preincubated with [32P]8N3-cADPR resulted in specific labeling of proteins of 140 and 100 kDa, which could be prevented by 8N3-cADPR or nanomolar concentrations of cADPR, but not by micromolar concentrations of ADP-ribose, AMP, ADP, ATP, cyclic AMP or inositol 1,4,5-trisphosphate. Caffeine, an agonist of Ca(2+)-induced Ca2+ release, preferentially inhibited the labeling of the 100 kDa as compared to the 140-kDa protein. These results suggest that cADPR may not interact directly with the ryanodine receptor, but may instead, exert its effect through intermediate proteins.