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After differential centrifugation of Ehrlich ascites cells by the de Duve method, NAD nucleosidase activities were found in the nuclear and the microsomal fractions, but not in the lysosomal fraction.The nuclear NAD nucleosidase is inhibited 500/, by 0.2 mM nicotinamide and is highly specific for /?-NAD. Its activity is markedly decreased after DNase treatment; it is associated with chromatin and is very probably identical with an enzyme that polymerizes the adenosine diphosphate ribose moiety of NAD to an acid-insoluble polymer first described in rat liver nuclei by Chambon, Weill, and Mandel. by 1.2 mM nicotinamide, and splits NAD, NADP and NMN a t a ratio of 1 : 0.5 : 0.2. NAD and NADP nucleosidase activities are reciprocally inhibited by NADP and NAD. Evidence is presented that the microsomal enzyme is active mainly a t the surface of the cell.Nicotinamide concentration may be one factor regulating the nuclear but not the microsomal NAD nucleosidase. Within the cell, NAD is protected by membrane structure from degradation by the microsomal NAD nucleosidase. The microsomal NAD nucleosidase is inhibited 50The activity of NAD nucleosidase (NADase) in homogenates of Ehrlich ascites cells far exceeds the calculated rate of NAD synthesis in intact cells [l]. The rapid decrease in the NAD content of ascites cells after treatment with alkylating agents [2,3] is very probably caused by a release of the intracellular inhibition of NADase [1,4]. The present study was undertaken to elucidate the mechanism(s) protecting NAD from degradation within the cell.