The effect of CO2 on the opening of stomata in the intact leaf of Commelina communis has been investigated. Full opening of stomatal apertures(around 18 μm) was achieved in the intact leaf by addition of CO2(900 μmol mol-1). At 90 minutes, the stomatal apertures of leaves treated with CO2 free air were reduced. In contrast, stomata opened most widely with the treatment of CO2 air at 90 minutes. The effects of light, CO2 air and CO2 free air on the change of membrane potential difference(PD) were measured. Fast hyperpolarization of guard cell membrane PD was recorded reaching up to -12 mV in response to light. If CO2 free air was given firstly, there was no response. When light was given after CO2 free air, the light effect was very clear. At the onset of CO2 air, the PD showed a dramatic hyperpolarization to about -25 mV. Changes in the pH of apoplast in intact leaves in response to CO2 air were observed. CO2 air caused a change of 0.4 pH unit. Therefore, it can be hypothesized that CO2 flowing could stimulate proton efflux which is a necessary precursor of stomatal opening.