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We used the natural abundance of stable isotopic ratios of hydrogen and oxygen in soil (0.05–3 m depth), plant xylem and precipitation to determine the seasonal changes in sources of soil water uptake by two native encroaching woody species (Pinus ponderosa P. & C. Lawson, Juniperus virginiana L.), and two C4 grasses (Schizachyrium scoparium (Michx.) Nash, Panicum virgatum L.), in the semiarid Sandhills grasslands of Nebraska. Grass species extracted most of their water from the upper soil profile (0.05–0.5 m). Soil water uptake from below 0.5 m depth increased under drought, but appeared to be minimal in relation to the total water use of these species. The grasses senesced in late August in response to drought conditions. In contrast to grasses, P. ponderosa and J. virginiana trees exhibited significant plasticity in sources of water uptake. In winter, tree species extracted a large fraction of their soil water from below 0.9 m depth. In spring when shallow soil water was available, tree species used water from the upper soil profile (0.05–0.5 m) and relied little on water from below 0.5 m depth. During the growing season (May–August) significant differences between the patterns of tree species water uptake emerged. Pinus ponderosa acquired a large fraction of its water from the 0.05–0.5 and 0.5–0.9 m soil profiles. Compared with P. ponderosa, J. virginiana acquired water from the 0.05–0.5 m profile during the early growing season but the amount extracted from this profile progressively declined between May and August and was mirrored by a progressive increase in the fraction taken up from 0.5–0.9 m depth, showing plasticity in tracking the general increase in soil water content within the 0.5–0.9 m profile, and being less responsive to growing season precipitation events. In September, soil water content declined to its minimum, and both tree species shifted soil water uptake to below 0.9 m. Tree transpiration rates (E) and water potentials (Ψ) indicated that deep water sources did not maintain E which sharply declined in September, but played an important role in the recovery of tree Ψ. Differences in sources of water uptake among these species and their ecological implications on tree–grass dynamics and soil water in semiarid environments are discussed.