The mechanisms for the production of hydrogen peroxide (H2O2) induced by abscisic acid (ABA) were investigated in suspension culture cells of tobacco BY-2 cells. The results showed that the immediate generation of H2O2, which was mainly derived from super-oxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA resulted in a time-dependent quick increase in plasma membrane (PM) NADPH oxidase activity, which coincided on time and magnitude with the elevation in ABA-induced accumulation of H2O2. Moreover, these enhanced effects were pronouncedly inhibited by two NADPH oxidase inhibitors, diphenylene iodonium and imidazole, suggesting that PM NADPH oxidase is involved in the rapid accumulation of H2O2 in cultured tobacco cells. In addition, analysis of the expression level of NtrbohD, a PM NADPH oxidase gene in tobacco, by RT-PCR and protein gel blot revealed that the gene at both mRNA and protein levels was upregulated by ABA, indicating that NtrbohD participates in the ABA-stimulated rapid production of H2O2 in tobacco culture cells. Taken together, these findings suggest that ABA induces the rapid accumulation of reactive oxygen species via NADPH oxidase in suspension culture cells of tobacco, and that NADPH oxidase and H2O2 appear to be important components in ABA signal transduction pathway in plants.