Abstract. 15Groundwater-dependent vegetation is globally distributed, having important ecological, social and economic value. Along with the groundwater resources upon which it depends, this vegetation is under increasing threat through excessive rates of groundwater extraction.In this study we examined one shallow-rooted and two deep-rooted tree species at multiple sites along a naturally occurring 20 gradient in depth-to-groundwater. We measured (i) stable isotope ratios of leaves (δ 13 C), xylem and groundwater (δ 2 H and δ 18 O); and (ii) leaf vein density. We established that foliar discrimination of 13 C (∆ 13 C) is a reliable indicator of groundwater use by vegetation and can also be used to estimate rooting depth. Through comparison with a continental-scale assessment of foliar ∆ 13 C, we also estimated the upper limits to annual rates of groundwater use. We conclude that maximum rooting depth for both deep-rooted species ranged between 9.4 m and 11.2 m and that annual rates of groundwater use ranged ca 25 1400 -1700 mm for Eucalyptus camaldulensis and 600 -900 mm for Corymbia opaca. Several predictions about hydraulic and leaf traits arising from the conclusion that these two species made extensive use of groundwater were supported by additional studies of these species in central Australia.