Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m(-2) UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including G alpha protein, hydrogen peroxide (H2O2), and nitric oxide (NO). UV-B triggered a significant increase in H2O2 or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H2O2 and NO generation are regulated by GPA1, the G alpha-subunit of heterotrimeric G proteins. UV-B-dependent H2O2 and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cG alpha). In addition, exogenously applied H2O2 or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cG alpha AtrbohD/AtrbohF and cG alpha nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that G alpha activation of NO production depends on H2O2. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H2O2 accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H2O2 production and subsequent Nia1-dependent NO accumulation.