In order to separate the net effect of growth at elevated [CO2] on stomatal conductance (gs) into direct and acclimatory responses, mid-day values of gs were measured for plants grown in field plots in open-topped chambers at the current ambient [CO2], which averaged 350 μmol mol−1 in the daytime, and at ambient + 350 μmol mol−1[CO2] for winter wheat, winter barley, potato and sorghum. The acclimatory response was determined by comparing gs measured at 700 μmol mol−1[CO2] for plants grown at the two [CO2]. The direct effect of increasing [CO2] from 350 to 700 μmol mol−1 was determined for plants grown at the lower concentration. Photosynthetic rates were measured concurrently with gs. For all species, growth at the higher [CO2] significantly reduced gs measured at 700 μmol mol−1[CO2]. The reduction in gs caused by growth at the higher [CO2] was larger for all species on days with low leaf to air water vapour pressure difference for a given temperature, which coincided with highest conductances and also the smallest direct effects of increased [CO2] on conductance. For barley, there was no other evidence for stomatal acclimation, despite consistent down-regulation of photosynthetic rate in plants grown at the higher [CO2]. In wheat and potato, in addition to the vapour pressure difference interaction, the magnitude of stomatal acclimation varied directly in proportion to the magnitude of down-regulation of photosynthetic rate through the season. In sorghum, gs consistently exhibited acclimation, but there was no down-regulation of photosynthetic rate. In none of the species except barley was the direct effect the larger component of the net reduction in gs when averaged over measurement dates. The net effect of growth at elevated [CO2] on mid-day gs resulted from unique combinations of direct and acclimatory responses in the various species.