Abaxial stomata from Vicia faba leaves grown in a growth chamber under constant light, temperature and humidity showed an elaborate pattern of aperture changes over the course of a light cycle. These aperture changes were tightly correlated with changes in chamber CO₂ concentration (r² =0.83). Changes in chamber [CO₂ ] resulted, in turn, from substantial daily fluctuations in ambient [CO₂ ], typical of the Los Angeles environment with a constant offset caused by photosynthesis and respiration of the plants within the chamber. The dominant role of the stomatal response to CO₂ in the control of aperture was confirmed by manipulation of chamber [CO₂ ]. Fast (15 min) increases and decreases in [CO₂ ] caused rapid decreases and increases in aperture, while constant |CO₂ ] resulted in constant aperture. In contrast, aperture changes in comparable plants grown under greenhouse conditions were tightly correlated with changes in incident solar radiation (r₂ =0.80), and poorly correlated with changes in [CO₂ ] (r₂ =0.09). Greenhouse‐grown plants transferred to growth chamber conditions showed no apparent response to CO₂ . These data indicate that growth‐chamber‐grown V, faba leaves provide an experimental system optimally suited for the study of the stomatal response to CO₂ , and suggest that acclimation to environmental conditions alters the sensitivity of stomata to CO₂ .