Significance: Nicotinamide adenine dinucleotide (NAD⁺) spans diverse roles in biology, serving as both an important redox cofactor in metabolism and a substrate for signaling enzymes that regulate protein post-translational modifications (PTMs).

Critical Issues: Although the interactions between these different roles of NAD⁺ (and its reduced form NADH) have been considered, little attention has been paid to the role of compartmentation in these processes. Specifically, the role of NAD⁺ in metabolism is compartment specific (e.g., mitochondrial vs. cytosolic), affording a very different redox landscape for PTM-modulating enzymes such as sirtuins and poly(ADP-ribose) polymerases in different cell compartments. In addition, the orders of magnitude differences in expression levels between NAD⁺-dependent enzymes are often not considered when assuming the effects of bulk changes in NAD⁺ levels on their relative activities.

Recent Advances: In this review, we discuss the metabolic, nonmetabolic, redox, and enzyme substrate roles of cellular NAD⁺, and the recent discoveries regarding the interplay between these roles in different cell compartments.

Future Directions: Therapeutic implications for the compartmentation and manipulation of NAD⁺ biology are discussed. Antioxid. Redox Signal. 31, 623–642.