The NAD glycohydrolase (NADase) was solubilized from intact erythrocytes with bacterial phosphatidylinositol-specific phospholipase C and purified to homogeneity by affinity chromatography on Cibacron blue-agarose. This purification procedure resulted in an approximately 85-fold purification with an overall recovery of 75%. The purified NADase has a molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 63,000 as determined by gel permeation column chromatography at pH 7.0. Two hybridoma cell lines secreting antibodies against NADase were established and the antibodies recognized the purified enzyme as well as a 65-kDa band from the extracts of rabbit erythrocyte ghost. The enzyme displayed a Km of 43 microM for beta-NAD, a Vmax of 23 mumol/min/mg, a broad pH optimum around pH 7.0, and pI of 5.0. Nicotinamide and isoniazid are inhibitors (Ki values, 2.5 and 3.5 mM, respectively) of the noncompetitive type. Adenosine diphosphoribose acts as a competitive inhibitor (Ki = 2.0 mM). Cibacron blue 3GA is a potent competitive inhibitor of NADase (Ki = 96 nM). The purified enzyme splits beta-NAD, NADP, and nicotinamide hypoxanthine dinucleotide among the compounds tested and does not exhibit transglycosidase activity. Amino acid composition of the rabbit erythrocyte enzyme differed from that of NADases of other species, and the purified NADase contains 8% carbohydrate and a stoichiometric amount of inositol.