C2H4 is associated with plant defense, but its role during the hypersensitive response (HR) remains largely uncharacterized. C2H4 production in tobacco (Nicotiana tabacum) following inoculation with HR-eliciting Pseudomonas syringae pathovars measured by laser photoacoustic detection was biphasic. A first transient rise (C2H4-I) occurred 1 to 4 h following inoculation with HR-eliciting, disease-forming, and nonpathogenic strains and also with flagellin (flg22). A second (avirulence-dependent) rise, at approximately 6 h (C2H4-II), was only seen with HR-eliciting strains. Tobacco leaves treated with the C2H4 biosynthesis inhibitor, aminoethoxyvinylglycine, suggested that C2H4 influenced the kinetics of a HR. Challenging salicylate hydroxylase-expressing tobacco lines and tissues exhibiting systemic acquired resistance suggested that C2H4 production was influenced by salicylic acid (SA). Disrupted expression of a C2H4 biosynthesis gene in salicylate hydroxylase tobacco plants implicated transcriptional control as a mechanism through which SA regulates C2H4 production. Treating leaves to increase oxidative stress or injecting with SA initiated monophasic C2H4 generation, but the nitric oxide (NO) donor sodium nitroprusside initiated biphasic rises. To test whether NO influenced biphasic C2H4 production during the HR, the NO synthase inhibitor NG-nitro-L-arginine methyl ester was coinoculated with the avirulent strain of P. syringae pv phaseolicola into tobacco leaves. The first transient C2H4 rise appeared to be unaffected by NG-nitro-L-arginine methyl ester, but the second rise was reduced. These data suggest that NO and SA are required to generate the biphasic pattern of C2H4 production during the HR and may influence the kinetics of HR formation.