1-(5-Phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride [2'-phospho-cyclic ADP-ribose, cAdo(2')P(5')PP-Rib] was prepared enzymatically from NADP+ using ADP-ribosyl-cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1-(5-phospho-beta-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cADP-Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo-(2')P(5')PP-Rib dose-dependently released Ca2+ from an intracellular, non-endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB. ALL T-lymphocytes. In contrast, the closely related compounds 1-(5-phospho-beta-D-ribosyl)3'phosphoadenosine 5'-phosphate cyclic anhydride and 1-(5-phospho-beta-D-ribosyl)cyclic 2',3'-phosphoadenosine 5'-phosphate cyclic anhydride did not induce Ca2+-release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2')P(5')PP-Rib partially overlapped with the Ca2+ pool sensitive to cADP-Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L. & Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+-release in T-lymphocyte cell lines, J. Immunol. 155, 3353-3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP-Rib preparation nor to catabolism of cAdo(2')P(5')PP-Rib to cADP-Rib.