The response of nocturnal stomatal conductance (gs,n) to rising atmospheric CO2 concentration ([CO2]) is currently unknown, and may differ from responses of daytime stomatal conductance (gs,d). Because night-time water fluxes can have a significant impact on landscape water budgets, an understanding of the effects of [CO2] and temperature on gs,n is crucial for predicting water fluxes under future climates. Here, we examined the effects of [CO2] (280, 400 and 640 mu mol mol-1), temperature (ambient and ambient + 4 degrees C) and drought on gs,n, and gs,d in Eucalyptus sideroxylon saplings. g s,n was substantially higher than zero, averaging 34% of gs,d. Before the onset of drought, gs,n increased by 85% when [CO2] increased from 280 to 640 mu mol mol-1, averaged across both temperature treatments. gs,n declined with drought, but an increase in [CO2] slowed this decline. Consequently, the soil water potential at which gs,n was zero (0) was significantly more negative in elevated [CO2] and temperature treatments. gs,d showed inconsistent responses to [CO2] and temperature. g s,n may be higher in future climates, potentially increasing nocturnal water loss and susceptibility to drought, but cannot be predicted easily from gs,d. Therefore, predictive models using stomatal conductance must account for both gs,n and gs,d when estimating ecosystem water fluxes.