The hypothesis that increases in cytosolic free calcium ([Ca2+]i) are a component of the CO2 signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca2+]i in response to challenge with 700 µl l−1 CO2. Elevated CO2 induced increases in guard cell [Ca2+]i which were similar to those previously reported in response to abscisic acid. [Ca2+]i returned to resting values following removal of the CO2 and further application of CO2 resulted in a second increase in [Ca2+]i. This demonstrated that the CO2-induced increases in [Ca2+]i were stimulus dependent. Removal of extracellular calcium both prevented the CO2-induced increase in [Ca2+]i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca2+ acts as a second messenger in the CO2 signal transduction pathway and that an increase in [Ca2+]i may be a requirement for the stomatal response to CO2.