Within Australia and globally there is considerable concern about the potential impacts of groundwater extraction on ecosystems dependent on groundwater. In this study we have combined heat pulse and isotopic techniques to assess groundwater use by riparian vegetation along the Daly River in the Northern Territory. The riparian forests of the Daly River exhibited considerable structural and floristic complexity. More than 40 tree species were recorded during vegetation surveys and these exhibited a range of leaf phonologies, implying complex patterns of water resource partitioning within the riparian forests. Water use was a function of species and season, and stand water use varied between 1.8 and 4.1 mm day^–1. In general, however, water use tended to be higher in the wet season than during the dry season, reflecting the contribution to stand water use by dry-season deciduous tree species. There was a strong relationship between stand basal area and stand water use in the wet season, but the strength of this relationship was lower in the dry season. The amount of groundwater use, as determined by analysis of deuterium concentrations in xylem sap, was principally a function of position in the landscape. Trees at lower elevations, closer to the river, used more groundwater than trees higher on the levees. By using a combination of techniques we showed that riparian vegetation along the Daly River was highly groundwater dependent and that these water-use requirements need to be considered in regional management plans for groundwater.