ADP-ribosyl cyclases (ADPRCs) are present from lower Metazoa to mammals and synthesize the Ca2+-active (di)nucleotides cyclic ADP-ribose (cADPR), NAADP(+), and ADP-ribose (ADPR), involved in the regulation of important cellular functions. NAADP(+) can be synthesized by ADPRCs from NADP(+) through a base-exchange reaction, which substitutes nicotinamide for nicotinic acid (NA). Here we demonstrate that ADPRCs from both lower and higher Metazoa (including human CD38) can also synthesize NAADP(+) starting from 2'-phospho-cyclic ADP-ribose (cADPRP) and NA. Comparison, on the two substrates cADPRP and NADP(+), of the relative rates of the reactions introducing NA and hydrolyzing/cyclizing the substrate, respectively, indicates that with all ADPRCs tested cADPRP is preferentially transformed into NAADP, while NADP(+) is preferentially cyclized or hydrolyzed to cADPRP/2'-pliosplio-ADP-ribose. cADPRP was detectable in retinoic acid-differentiated, CD38(+) HL-60 cells, but not in Undifferentiated, CD38(-) cells. These results suggest that cADPRP may be a NAADP(+) precursor in ADPRC(+) cells. (c) 2006 Elsevier Inc. All rights reserved.