The internal conductance to CO2 supply from substomatal cavities to sites of carboxylation poses a large limitation to photosynthesis. It is known that internal conductance is decreased by soil water deficits, but it is not known if it is affected by atmospheric water deficits (i.e. leaf to air vapour pressure deficit, VPD). The aim of this paper was to examine the responses of internal conductance to atmospheric and soil water deficits in seedlings of the evergreen perennial Eucalyptus regnans F. Muell and the herbaceous plants Solanum lycopersicum (formerly Lycopersicon esculentum) Mill. and Phaseolus vulgaris L. Internal conductance was estimated with the variable J method from concurrent measurements of gas exchange and fluorescence. In all three species steady-state stomatal conductance decreased by approximately 30% as VPD increased from 1 kPa to 2 kPa. In no species was internal conductance affected by VPD despite large effects on stomatal conductance. In contrast, soil water deficits decreased stomatal conductance and internal conductance of all three species. Decreases in stomatal and internal conductance under water deficit were proportional, but this proportionality differed among species, and thus the relationship between stomatal and internal conductance differed among species. These findings indicate that soil water deficits affect internal conductance while atmospheric water deficits do not. The reasons for this distinction are unknown but are consistent with soil and atmospheric water deficits having differing effects on leaf physiology and/or root-shoot communication.