Human CD38 is an ectoenzyme expressed on the surface of B-cells that makes cyclic-ADP-ribose (cADPR) and ADP-ribose from NAD(+) (nicotinamide diphosphate ribose, oxidized form). The compound cADPR is a potent second messenger for calcium release inside cells. Nicotinamide guanine dinucleotide (NGD(+)) is also cyclized by CD38 to form cGDPR (cyclic guanosine diphosphate ribose) and hydrolyzed to form GDPR (guanosine diphosphate ribose). Kinetic isotope effect studies in the presence of 20 mM nicotinamide gave 1'-H-3 and 1'-C-14 isotope effects of 1.02 +/- 0.01 and 1.00 +/- 0.01, respectively, for the cyclization reaction and 1.23 +/- 0.01 and 1.02 +/- 0.01, respectively, for the hydrolysis reaction. These values support a covalent intermediate. The existence of a covalent intermediate was established by reaction of ara-F-NMN+ (arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide) with the enzyme. This compound reacted to release 1 mol of nicotinamide/mole of CD38 monomer add to form an inactive covalent intermediate. Reaction with excess nicotinamide rescued catalytic activity with an apparent K-m of 17 +/- 5 mM and a V-max of 0.023 +/- 0.003 s(-1). Proof of covalent labeling of the enzyme by this inhibitor was obtained by MS analysis. Treatment of CD38 with ara-F-NMN+ increased mass by 215 amu, consistent with formation of CD38-fluoro-sugar monophosphate. Tryptic digestion in urea, phosphatase treatment, and purification of peptides in combination with MALDI-PSD permitted identification of Glu226 as the amino acid nucleophile. This residue is highly conserved across all ADP-ribosyl (adenosine diphosphate ribosyl) cyclases. The covalent intermediate inherent to the catalytic mechanism of human CD38 provides chemical precedent for related NAD(+) glycosyltransferases.