In the present study the response of stomatal conductance (gs) to increasing leaf-to-air vapour pressure difference (D) in early season C 3 (Bromus japonicus) and late season C4 (Bothriochloa ischaemum) grasses grown in the field across a range of CO2 (200-550 μmol mol-1) was examined. Stomatal sensitivity to D was calculated as the slope of the response of gs to the natural log of externally manipulated D (dgs/dlnD). Increasing D and CO2 significantly reduced gs in both species. Increasing CO2 caused a significant decrease in stomatal sensitivity to D in Br. japonicus, but not in Bo. ischaemum. The decrease in stomatal sensitivity to D at high CO2 for Br. japonicus fit theoretical expectations of a hydraulic model of stomatal regulation, in which gs varies to maintain constant transpiration and leaf water potential. The weaker stomatal sensitivity to D in Bo. ischaemum suggested that stomatal regulation of leaf water potential was poor in this species, or that non-hydraulic signals influenced guard cell behaviour. Photosynthesis (A) declined with increasing D in both species, but analyses of the ratio of intercellular to atmospheric CO2 (Ci/Ca) suggested that stomatal limitation of A occurred only in Br. japonicus. Rising CO2 had the greatest effect on gs and A in Br. japonicus at low D. In contrast, the strength of stomatal and photosynthetic responses to CO2 were not affected by D in Bo. ischaemum. Carbon and water dynamics in this grassland are dominated by a seasonal transition from C3 to C4 photosynthesis. Interspecific variation in the response of gs to D therefore has implications for predicting seasonal ecosystem responses to CO2.