Systems for exposing plants to elevated concentrations of CO2 may expose plants to pulses of CO2 concentrations considerably above the control set point. The importance of such pulses to plant function is unknown. Single leaves of wheat (Triticum aestivum cv Choptank) and rice (Oryza sativa cv Akitakomachi) were exposed for 30 minutes to pulses of elevated CO2 similar to the frequency, duration and magnitude of pulses observed in free-air CO2 enrichment systems. Stomatal conductance decreased within a few minutes of exposure to once per minute pulses of high CO2 of all the durations tested, in both species. Both species had 20–35% lower stomatal conductance for at least 30 min after the termination of the pulses. After the pulses had stopped, in all cases photosynthesis was below the values expected for the observed substomatal CO2 concentration, which suggests that either patchy stomatal closure occurred or that photosynthesis was directly inhibited. It was also found that a single, 2 s pulse of elevated CO2 concentration reaching a maximum of 1000 µmol mol−1 decreased stomatal conductance in both species. On the basis of these results, it is probable that plants in many CO2 enrichment systems have lower photosynthesis and stomatal conductance than would plants exposed to the same mean CO2 concentration but without pulses of higher concentration.