There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H2O2 as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H2O2 and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H2O2 accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H2O2 in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H2O2in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H2O2. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H2O2 comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues