Abstract Transpiration of and water sources for Eucalyptus largiflorens F. Muell trees at three sites on a semi-arid floodplain of the lower River Murray in southern Australia were investigated during the course of a flood. Two of the sites were flooded for 32 days while the third was not flooded. At both flooded sites transpiration was not suppressed during the period when the sites were flooded, indicating that anoxia did not occur. Available literature suggests that E. largiflorens has very low O2 consumption, and that lateral roots can absorb O2 from soil water at very low partial pressures. The oxygen stored in the unsaturated region of the soil appeared sufficient, in this case, to meet the requirements of the trees. Transpiration at the flooded sites did not increase in comparison with that at the non-flooded site in the period following flooding. We suggest that this was possibly due to the above-average rainfall experienced in the months preceding the flood and/or physiological adaptations to the highly saline conditions that prevented the trees taking full advantage of the additional water. The sources of water used by the trees were investigated using the naturally occurring stable isotopes of water and measurements of soil water suction. These showed that prior to the flood shallow rain-derived soil water was being used at all three sites. Ten days after flooding ceased trees at both flooded sites were using shallow flood-derived soil water while at the non-flooded sites they were using groundwater. Results from the flooded sites 7 and 22 weeks after flooding ceased showed that as the flood- and rain-derived water became limited the trees supplemented their supplies with groundwater from the capillary fringe. The non-flooded trees used various combinations of rain-derived soil water and groundwater during this period.