A rapid and localized programmed cell death – the hypersensitive response (HR) – is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H2O2 generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H2O2 generation following challenge with avirulent strains of Pseudomonas syringae pv. (P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H2O2-responsive transgene AoPR10-GUS, the dually responsive (H2O2 and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10- and PR1a-GUS expression and cell death. Conversely, varying H2O2 generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci, which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H2O2 generation by an unknown mechanism.