Active oxygen species (AOS) are central components of the defence reactions of plants against pathogens. Plant respiratory burst oxidase homologues (RBOH) of gp91(phox), a plasma membrane protein of the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, play a prominent role in AOS production. The role of two RBOH from Nicotiana benthamiana, NbrbohA and NbrbohB that encode plant NADPH oxidase in the process of elicitor-induced stomatal closure and hypersensitive cell death is described here. NbrbohA was constitutively expressed at a low level, whereas NbrbohB was induced when protein elicitors exist (such as boehmerin, harpin, or INF1). The virus-induced gene-silencing (VIGS) method was used to produce single-silenced (NbrbohA or NbrbohB) and double-silenced (NbrbohA and NbrbohB) N. benthamiana plants. The hypersensitive response (HR) of cell death and pathogenesis-related (PR) gene expression of these gene-silenced N. benthamiana plants, induced by various elicitors, are examined. The HR cell death and transcript accumulation of genes related to the defence response (PR1) were slightly affected, suggesting that RBOH are not essential for elicitor-induced HR and activation of these genes. Interestingly, gene-silenced plants impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production, but not elicitor-induced cytosolic calcium ion accumulation and elicitor-triggered AOS production in guard cells. These results indicate that RBOH from N. benthamiana function in elicitor-induced stomatal closure, but not in elicitor-induced HR.