• The response of nocturnal stomatal conductance (g s,n) to rising atmospheric CO 2 concentration ([CO 2]) is currently unknown, and may differ from responses of daytime stomatal conductance (g s,d). Because night-time water fluxes can have a significant impact on landscape water budgets, an understanding of the effects of [CO 2] and temperature on g s,n is crucial for predicting water fluxes under future climates. • Here, we examined the effects of [CO 2] (280, 400 and 640μmolmol -1), temperature (ambient and ambient+4°C) and drought on g s,n, and g s,d in Eucalyptus sideroxylon saplings. • g s,n was substantially higher than zero, averaging 34% of g s,d. Before the onset of drought, g s,n increased by 85% when [CO 2] increased from 280 to 640μmolmol -1, averaged across both temperature treatments. g s,n declined with drought, but an increase in [CO 2] slowed this decline. Consequently, the soil water potential at which g s,n was zero (Ψ 0) was significantly more negative in elevated [CO 2] and temperature treatments. g s,d showed inconsistent responses to [CO 2] 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 g s,d. Therefore, predictive models using stomatal conductance must account for both g s,n and g s,d when estimating ecosystem water fluxes.