The hydrogen isotopic signature (δ²H) of water in trees contains information on plant functional responses to climatic changes and on the origin of the water. This is also true for the non-exchangeable hydrogen isotopic signature (δ²H_NE) of plant organic matter, which contains additional physiological and biochemical information that can be dated to specific years if extracted from annual rings of trees. Despite this potential for gaining unique insights from δ²H_NE of tree-ring cellulose (δ²H_TRC), it has not been widely used compared to other isotope signals, likely due to challenging methodological constraints and interpretations of these isotopic signals. In this chapter, we first summarize hydrogen isotope (²H-) fractionation that occurs between source water and tree rings and review methods (e.g. nitration, equilibration, position-specific applications) and calculations to determine δ²H_NE in tree material. Building upon a summary of the current state of knowledge, this chapter also provides an exhaustive synthesis of δ²H_TRC papers, applications, and associated data from approximately 180 sites across the globe (paired with modelled precipitation δ²H values and climate data). The data allow us to investigate the hydrological-climatic effects driving δ²H_TRC pattern on a global scale, the relationship of hydrogen with oxygen isotopes in the same tree-ring material, as well as the influence of physiological-biochemical effects (e.g., species differences, tree growth) that appear to be more important on local or temporal scales  than on a large spatial scales. Thus, when local hydro-climatic influences on source water δ²H can be isolated, δ²H_TRC gives novel insights on tree physiological responses to abiotic and biotic stresses. We conclude that the growing constellation of tree-ring metrics, including advancements in ²H-processing (i.e., equilibration techniques allowing rapid determinations of δ²H_NE) and further refinements to the understanding of post-photosynthetic ²H-fractionations will together provide many new opportunities to understand past climates and ecophysiology by using δ²H in tree rings.