This study investigated the influence of meteorological, pedospheric and physiological factors on the water relations of Scots pine, as characterized by the origin of water taken up, by xylem transport as well as by carbon isotope discrimination (Δ13C) and oxygen isotope enrichment (Δ18O) of newly assimilated organic matter. For more than 1 year, we quantified δ2H and δ18O of potential water sources and xylem water as well as Δ13C and Δ18O in twig and trunk phloem organic matter biweekly, and related these values to continuously measured or modelled meteorological parameters, soil water content, stand transpiration (ST) and canopy stomatal conductance (Gs). During the growing season, δ18O and δ2H of xylem water were generally in a range comparable to soil water from a depth of 2–20 cm. Long residence time of water in the tracheids uncoupled the isotopic signals of xylem and soil water in winter. Δ18O but not Δ13C in phloem organic matter was directly indicative of recent environmental conditions during the whole year. Δ18O could be described applying a model that included 18O fractionation associated with water exchange between leaf and atmosphere, and with the production of organic matter as well as the influence of transpiration. Phloem Δ13C was assumed to be concertedly influenced by Gs and photosynthetically active radiation (PAR) (as a proxy for photosynthetic capacity). We conclude that isotope signatures can be used as effective tools (1) to characterize the seasonal dynamics in source and xylem water, and (2) to assess environmental effects on transpiration and Gs of Scots pine, thus helping to understand and predict potential impacts of climate change on trees and forest ecosystems.