Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5' coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using p-nitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.