Abstract NAD + catabolism in rabbit reticulocyte lysates has been studied using [ adenine- 14C]-, [ carbonyl- 14C]- and [ pyrophosphate- 32P]NAD +. NAD + is rapidly and quantitatively converted to ATP and ribose 5-phosphate. The ATP is formed via ADPribose. NAD + glycohyrolase (NADase), ADPribose pyrophosphatase, adenylate and/or phosphoglycerate kinase, and pyruvate kinase are essential in the conversion of NAD + to ATP. This is based on the following four experiments. First, theobromine and nicotinamide, inhibitors of NADase, prevent the conversion of NAD + to ATP. Exogenously supplied NADase results in the accumulation of ADPribose in 1–2 min. The ADPribose is then rapidly converted to ATP. Second, incubation of NAD + with dialyzed lysate results in the accumulation of AMP. Third, in the presence of P 1,P 5-di(adenosine-5′)-pentaphosphate, an inhibitor of adenylate kinase, the decrease in ATP formation parallels the increase in AMP. Fourth, [ pyrophosphate- 32P]NAD + is converted to [ 32P]ATP and ribose 5-[ 32P]phosphate. The ribose 5-[ 32P]phosphate formed from the catabolism of [ pyrophosphate- 32P]NAD + is not further utilized in these lysate preparations. The hydrolysis of NAD + and ADPribose is catalyzed by NAD + glycohydrolase and ADPribose pyrophosphatase. These two enzymes have been resolved by Sephadex G-100 gel filtration and have distinctly different (i) pH profiles (ii) temperature-activity profiles and (iii) activities with NAD + analogs.