1. Stable isotope ratios of water (δ 18 O, δ 2 H) have long been used to study a core question in plant ecology and ecohydrology: 'From where do plants take up water?' Indeed, decades of research has involved sampling potential plant water sources in the subsurface, classifying those sources as distinct endmembers (e.g. deep vs. shallow soil waters) and then evaluating their contributions to a xylem water sample through mixing-model analysis to identify the depths of root water uptake. 2. However, more detailed interrogations of the subsurface and plant domains have revealed under-considered transport and isotopic fractionation phenomena. These now apparent complexities raise new questions and challenge the many past assumptions inherent in endmember-mixing models that now seem overly simple. 3. Here, we introduce discussions of these recent insights and provide an overview of isotope effects that occur naturally in the root zone and in the plant, as well as artificially during sample handling. Better accounting for these complexities and their associated uncertainties can lead to more accurate and robust study designs, analytical frameworks and, ultimately, inferences. 4. Finally, to more robustly characterize plant water sources using δ 18 O and δ 2 H, we provide some practical recommendations that aim at maximizing the isotopic contrast between endmembers and/or minimizing potential uncertainties. K E Y W O R D S ecohydrology, endmember-mixing models, plant water uptake, review, root zone, stable water isotopes, uncertainties 1 | INTRODUC TION AND MOTIVATION Plant water use can represent up to 90% of terrestrial evapotranspiration (Jasechko et al., 2013), and therefore is an important driver of the global water cycle (Sellers et al., 1997). Because plant water relations are closely linked to carbon and nutrient relations (Schulze, 1982), understanding plant water supplies is also key to predict global carbon and nutrient cycles (Lange, Kappen, | 1353 Methods in Ecology and Evoluঞon von FREYBERG Et al. How to cite this article: von Freyberg J, Allen ST, Grossiord C, Dawson TE. Plant and root-zone water isotopes are difficult to measure, explain, and predict: Some practical recommendations for determining plant water sources.