Although hydraulic redistribution has been observed for a range of tree species, including Eucalyptus kochii subsp. borealis (C. Gardner) D. Nicolle, there is limited direct evidence that water taken up by deep roots in moist soil is in fact exuded by shallow roots in dry soil. This paper reports an experiment designed to test this hypothesis. Water enriched with deuterium was added to the groundwater via a slotted tube at 4.5 m depth below 5-year-old E. kochii subsp. borealis trees. Nocturnal sap flow increased markedly immediately after deep irrigation, indicating that the trees were using water from this depth. Two weeks later, samples of surface soil and xylem water were found to contain levels of deuterium up to 30% higher than soils and xylem water from a control plot upslope of the main treatment plot. This is strong evidence that trees used groundwater and that efflux of important amounts of hydraulically redistributed water occurred via the roots of E. kochii subsp. borealis.