The production of reactive oxygen species (ROS) is part of the defence reaction of plants against invading pathogens. The effect of ROS on filamentous fungi is still unclear. In this study, ratiometric redox-sensitive green fluorescent protein (roGFP) was introduced as a tool for in vivo measurement of the cellular redox status in filamentous fungi. A fungal expression system for roGFP2 was constructed. Expressed in Botrytis cinerea, roGFP2 reversibly responded to redox changes induced by incubation with H2O2 or dithiothreitol, which was determined by confocal laser scanning microscopy imaging and fluorometry. As the sensor detects the redox potential of the cellular glutathione pool, it was used to analyse the kinetics of GSH (glutathione, reduced form) recovery after H2O2 treatment. The transcription factor Bap1 is the main transcriptional regulator of H2O2-scavenging proteins in B. cinerea. When compared with the wild-type, GSH recovery in the Δbap1 deletion mutant was affected after repeated H2O2 treatment. ROS and intracellular redox changes can be used by fungi for signalling purposes. In planta experiments, performed in this study, indicated that redox processes seem to be important for the differentiation of penetration structures. During the penetration of onion epidermal cells, the status of the cellular glutathione pool differed between appressoria-like structures and infecting hyphae, being reduced in the presence of infecting hyphae and more oxidized around appressoria-like structures.