Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 mol m Ϫ2 s Ϫ1 , red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 mol m Ϫ2 s Ϫ1 . Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 mol m Ϫ2 s Ϫ1 ) to a 100 mol m Ϫ2 s Ϫ1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 mol m Ϫ2 s Ϫ1 red light, 10 mol m Ϫ2 s Ϫ1 blue light opened stomata in both WT and npq1 mutant stomata but not in the phot1 phot2 double mutant. In npq1, blue light-stimulated opening was reversed by far-red but not green light, indicating that npq1 has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 mol m Ϫ2 s Ϫ1 blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.Many steps in the sensory-transducing cascade mediating blue light-specific stomatal movements are well characterized (Assmann, 1993). Guard cells are turgor valves that control the dimensions of the stomatal pore by changes in water content caused by changes in their osmotic potential. Blue light-specific stomatal opening is mediated by potassium and chloride uptake and malate biosynthesis. Ion uptake is driven by an electrochemical gradient generated by a proton-pumping ATPase activated by blue light (Tallman, 1992). The activation of the ATPase is mediated by the phosphorylation of its C terminus by a Ser/Thr kinase, facilitated by the binding of a 14-3-3 protein (Kinoshita and Shimazaki, 1999). Blue lightspecific stomatal opening has an action spectrum, showing a maximum at 450 nm and two minor peaks at 420 and 470 nm (Karlsson, 1986).Genetic, biochemical, and physiological studies have identified the chloroplastic carotenoid, zeaxanthin, as a blue light photoreceptor in guard cells (Frechilla et al., 1999). Two recent studies, however, have reported that stomata from the zeaxanthin-less Arabidopsis mutant, npq1, open in response to blue light (Eckert and Kaldenhoff, 2000;Kinoshita et al., 2001) and questioned the validity of the zeaxanthin hypothesis. One of these studies further reported that stomata from the phot1 phot2 double mutant of Arabidopsis lack a blue light response and hypothesized that phototropin is the main photoreceptor mediating blue light responses in guard cells (Kinoshita et al., 2001).The photobiological properties of guard cells complicate the analysis of stomatal opening in response to blue light (Lasceve et al., 1999)...