Leaves were collected from 21 different grapevine (Vitis ssp.) genotypes with varying resistance to powdery mildew disease caused by Uncinula necator. For leaves collected from field-grown vines in spring there was a correlation between resistance rating and activity of chitinase and β-1,3-glucanase. The correlation was greater with the sum of the two enzyme activities. In contrast, no correlation was obtained for leaves collected during summer. With leaves from glasshouse grown vines, wounding or infection with powdery mildew increased both chitinase and β-1,3-glucanase activity. Light microscope examination of detached leaves inoculated with U. necator conidia showed that germination appeared to occur at the same rate on leaves of a susceptible (Sultana) and a resistant (Seyval) genotype. Subsequent development of mycelia was severely restricted on the resistant genotype but it was prolific on the sensitive genotype. A bioassay was developed based on germination and extension of the germ tube of U. necator conidia on agar plates. Agar preparations containing desalted crude extracts of grapevine leaves inhibited growth and caused the tips of the hyphae to rupture. The effect was not observed with boiled extracts and was greater with extracts from resistant genotypes. Chitinase and β-1,3-glucanase were purified 760-fold and 46-fold respectively from leaves of Seyval grapevines. The purified enzyme preparations inhibited germ tube growth, with the effect being more prominent in the presence of both enzymes. The results demonstrate that these two enzymes have antifungal activity against U. necator, and are consistent with these pathogenesis-related proteins having a role in defence of grapevines against powdery mildew.