We investigated how leaf hydraulic conductance (Kleaf) of loblolly pine trees is influenced by soil nitrogen amendment (N) in stands subjected to ambient or elevated CO2 concentrations (CO2a and CO2e, respectively). We also examined how Kleaf varies with changes in reference leaf water potential (Ψleaf-ref) and stomatal conductance (gs-ref) calculated at vapour pressure deficit, D of 1 kPa. We detected significant reductions in Kleaf caused by N and CO2e, but neither treatment affected pre-dawn or midday Ψleaf. We also detected a significant CO2e-induced reduction in gs-ref and Ψleaf-ref. Among treatments, the sensitivity of Kleaf to Ψleaf was directly related to a reference Kleaf (Kleaf-ref computed at Ψleaf-ref). This liquid-phase response was reflected in a similar gas-phase response, with gs sensitivity to D proportional to gs-ref. Because leaves represented a substantial component of the whole-tree conductance, reduction in Kleaf under CO2e affected whole-tree water use by inducing a decline in gs-ref. The consequences of the acclimation of leaves to the treatments were: (1) trees growing under CO2e controlled morning leaf water status less than CO2a trees resulting in a higher diurnal loss of Kleaf; (2) the effect of CO2e on gs-ref was manifested only during times of high soil moisture.