Abstract A gene was cloned from Botrytis cinerea that encodes a protein homologous to glutathione S-transferase (GST). The gene, denominated Bcgst1, is present in a single copy and represents the first example of such a gene from a filamentous fungus. The biochemical function of GSTs is to conjugate toxic compounds to glutathione, thereby detoxifying the compound. In many other organisms, GST plays a role in chemical stress tolerance. We anticipated that GST functions for B. cinerea as a potential virulence factor, enabling the fungus to tolerate fungitoxic plant defence compounds. The expression of Bcgst1 mRNA under various presumably stressful conditions was investigated. Bcgst1 mRNA is expressed at a basal level in liquid cultures and is induced upon addition of hydrogen peroxide to the medium. The level of Bcgst1 mRNA expression during infection of tomato leaves parallels the level of actin mRNA. The role of the Bcgst1 gene in the virulence of Botrytis cinerea was evaluated by constructing gene disruption mutants. Three independent disruption mutants were obtained. The virulence of two mutants on tomato leaves was evaluated. Neither of the mutants showed a decrease in virulence, indicating that the Bcgst1 gene is not essential for virulence on tomato leaves under the conditions tested.