In the Japanese pear pathotype of Alternaria alternata, H2O2 is generated solely from penetration pegs and not from other portions of subcuticular hyphae within the pectin layers of host leaves. A close association between H2O2 generation and fungal aggressiveness is expected because the pegs are important for fungal penetration into the host epidermis. To determine the potential role of reactive oxygen species in microbial pathogenicity, we studied the inhibitory effects of the antioxidant reagent ascorbic acid and the NADPH oxidase inhibitor diphenylene iodonium on infection of the pathogen. In our study, we showed H2O2 generation to be inhibited by inoculation with the mixture of ascorbic acid or diphenylene iodonium and spores at the pegs in the spore-inoculated host leaves. The decrease of generation in the pegs resulted in penetration failure, indicating that H2O2 generation probably contributed to strengthening of the penetration and probably was recruited by NADPH oxidase.