Stomatal conductance was quantified with sap flux sensors and whole-tree chambers in mature Norway spruce (Picea abies (L.) Karst.) trees after 3 years of exposure to elevated CO2 concentration ([CO2]) in a 13-year nutrient optimization experiment. The long-term nutrient optimization treatment increased tree height by 3.7 m (67%) and basal diameter by 8 cm (68%); the short-term elevated [CO2] exposure had no effect on tree size or allometry. Nighttime transpiration was estimated as ∼7% of daily transpiration in unchambered trees; accounting for the effect of nighttime flux on the processing of sap flux signals increased estimated daily water uptake by ∼30%. Crown averaged stomatal conductance (gs) was described by a Jarvis-type model. The addition of a stomatal response time constant (τ) and total capacitance of stored water (Ctot) improved the fit of the model. Model estimates for Ctot scaled with sapwood volume of the bole in fertilized trees. Hydraulic support—defined as a lumped variable of leaf-specific hydraulic conductivity and water potential gradient (KlΔΨ) —was estimated from height, sapwood-to-leaf area ratio (As:Al) and changes in tracheid dimensions. Hydraulic support explained 55% of the variation in gs at reference conditions for trees across nutrient and [CO2] treatments. Removal of ∼50% of Al from three trees yielded results suggesting that stomatal compensation (i.e., an increase in gs) after pruning scales inversely with KlΔΨ, indicating that the higher the potential hydraulic support after pruning, the less complete the stomatal compensation for the increase in As:Al.