In the Rio Grande Plains of southern Texas, subtropical thorn woodland has replaced relatively open grassland/savanna during the past 200 years. To investigate potential changes in the hydrologic cycle of this system, the authors determined the natural abundance of 2H and 18O in rainfall, soil water and plant water in three habitats representing a successional chronosequence in the transition from open grassland to closed canopy woodland. Precipitation was isotopically identical with groundwater and fell on the meteoric water line with a weighted mean δ2H of -22per mill and a weighted mean δ18O of -4.3per mill. δ2H and δ18O of precipitation did not vary seasonally, but showed a significant 'amount effect'. Correlations between δ2H and δ18O of soil water indicated higher evaporation rates in wooded areas than in grassland. It is suggested that this result does not reflect higher evaporation rates, but rapid drying of the soil via transpiration followed by kinetic fractionation associated with evaporation in dry soils. δ2H and δ18O of plant and soil water indicated that more recently established woody plants associated with earlier successional stages had shallower root systems and obtained water from the upper 150 cm of the soil profile, while those associated with later successional stages had deeper root systems and acquired water below that depth. By reducing transpirational leaf area and root biomass in the grass layer, long term grazing in this former grassland may have enabled deeper infiltration of soil water, creating an opportunity for development of a community dominated by more deeply rooted woody plant species capable of exploiting this deeper water resource.