ATP-binding cassette (ABC) transporters are a large superfamily of proteins responsible for the ATP-dependent translocation of a wide variety of different en-dogenous and exogenous toxicants across cellular membranes. We used a PCR-based approach to isolate from Botrytis cinerea a DNA fragment that proved be an ABC type transporter gene following sequencing and Southern blot analyses. This gene, named BcatrO, was characterized by RT-PCR expression and knock-out mutant phenotype analysis. Treatment of B. cinerea with H2O2 and methyl viologen enhanced transcription of BcatrO. An increase of the gene transcripts was also observed in the early stages of pathogenesis after inoculation of the fungus to bean leaves. BcatrO knock-out mutants of B. cinerea were more sensitive than the wild type both to H2O2 and fungicide fenpiclonil, but displayed the same virulence. The possible role of BcatrO in a mechanism of resistance of B. cinerea to reactive oxygen species (ROS) or their products and in pleiotropic drug resistance phenomena is discussed.