The numerous biological roles of NAD+ are organized and coordinated via its compartmentalization within cells. The spatial and temporal partitioning of this intermediary metabolite is intrinsic to understanding the impact of NAD+ on cellular signaling and metabolism. We review evidence supporting the compartmentalization of steady-state NAD+ levels in cells, as well as how the modulation of NAD+ synthesis dynamically regulates signaling by controlling subcellular NAD+ concentrations. We further discuss potential benefits to the cell of compartmentalizing NAD+, and methods for measuring subcellular NAD+ levels. NAD+ serves an essential role as an electron acceptor (via hydride transfer) in central carbon metabolism. In the absence of intracellular NAD+, cells cannot produce ATP. However, even moderate diminishments in NAD+ levels can limit the signaling activity of NAD+-consuming enzymes. NAD+ concentrations differ in different parts of the cell, and there are distinct subcellular requirements for NAD+. Dynamic modulation of subcellular, and possibly extracellular, NAD+ concentrations represent an emerging mechanism for regulating specific NAD+-dependent pathways. Compartmentalization of NAD+ helps to time responses, communicate cellular status, and protect crucial NAD+ pools. Genetically encoded sensors represent promising approaches for additional development to generate a molecular toolbox for measuring and studying fluctuations in levels of compartmentalized NAD+. [ABSTRACT FROM AUTHOR]