SummaryUnderstanding isotope fractionation mechanisms is fundamental for analyses of plant ecophysiology and paleoclimate based on tree-ring isotope data.To provide new insights into isotope fractionation, we analysed intramolecular13C discrimination in tree-ring glucose (Δi’,i= C-1 to C-6) and metabolic deuterium fractionation at H1and H2(εmet) combinedly.This dual-isotope approach enabled deconvolution of isotope signals. We found evidence for physiological processes affectingΔ1’ andΔ3’ which respond to air vapour pressure deficit (VPD), and processes affectingΔ1’,Δ2’, andεmetwhich respond to precipitation but notVPD. These relationships exhibit change points dividing a period of homeostasis (1961-1980) from a period of metabolic adjustment (1983-1995). Homeostasis may result from sufficient groundwater availability. Additionally, we foundΔ5’ andΔ6’ relationships with radiation and temperature which are temporally stable and consistent with previously proposed isotope fractionation mechanisms.Based on the multitude of climate covariables, intramolecular carbon isotope analysis has an extraordinary potential for climate reconstruction. While isotope fractionation beyond leaves is usually considered to be constant, we propose significant parts of the carbon and hydrogen isotope variation in tree-ring glucose originate in stems (precipitation-dependent signals). As basis for follow-up studies, we propose mechanisms introducingΔ1’,Δ2’,Δ3’, andεmetvariability.