We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicais, and (b) H202. Both methyl viologen and H 2 0 2 inhibited stomatal opening and promoted stomatal closure. At concentrations 510-5 M, the effects of methyl viologen and H 2 0 2 on stomatal behavior were reversible and were abolished by 2 mM ECTA or 10 PM verapamil. I n addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by ECTA or verapamil, methyl viologen and H 2 0 2 caused an increase in guard cell cytosolic free Ca2+ ([Ca'+l,), which was abolished in the presence of ECTA. Therefore, at low concentrations of methyl viologen and H202, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatai aperture and the associated increases i n [Ca2+],. This suggests that in this concentration range the effects of the treatments are Ca'+-dependent and are mediated by changes in [Ca2+],. I n contrast, at concentrations of methyl viologen and H,O, >10-5 M, ECTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H202 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+Ii and the possible disruption of guard-cell ca'+ homeostasis are discussed in relation t o the processes of Ca'+-based signal transduction in stomatal guard cells and the control of stomatal aperture.