Active oxygen species (AOS) are responsible for triggering defense responses in plants. Respiratory burst oxidase homologs (rboh genes) have been implicated in AOS generation. We have isolated two rboh cDNAs, NbrbohA and NbrbohB, from Nicotiana benthamiana leaves. NbrbohA was expressed constitutively at a low level and the transcripts were increased after mechanical stress of control leaf infiltration, whereas NbrbohB was induced specifically by the protein elicitor INF1 from the potato pathogen Phytophthora infestans . We examined the function of the Nbrboh genes in AOS generation and in the hypersensitive response (HR) using virus-induced gene silencing (VIGS). VIGS indicated that both genes are required for H₂O₂ accumulation and for resistance to Phytophthora. VIGS of Nbrboh genes also led to a reduction and delay of HR cell death caused by INF1. We further demonstrate that the induction of HR-like cell death by overexpression of a constitutively active mutant of a mitogen-activated protein kinase kinase, MEK^DD, is compromised by VIGS of NbrbohB . We found that MEK^DD induced NbrbohB but not NbrbohA . This work provides genetic evidence for the involvement of a mitogen-activated protein kinase cascade in the regulation of rboh genes.