Nonspecific penetration resistance due to papilla formation and race-specific hypersensitive response (HR) can both contribute to Blumeria graminis resistance in barley. Some effective papillae form even in the susceptible cv. Pallas and the isoline P01 carries the additional Mla1 allele conditioning HR. The NO-specific stain DAF-2DA (4,5-diaminofluorescein-2-diacetate) revealed a transient NO generation burst commencing 10 h after inoculation (h.a.i.) in close association with sites of papilla formation in both barley lines. In P01 a burst of NO production throughout some attacked cells was initiated around 10–12 h.a.i. and this preceded whole-cell autofluorescence indicative of HR. The specificity of DAF-2DA staining was demonstrated by the suppression of staining following application of the NO scavenger C-PTIO (1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide). In addition, C-PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla-based resistance. Furthermore, C-PTIO application slightly delayed HR in P01 whereas, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated HR in P01 and increased cell death frequency in Pallas. Thus, NO generation is one of the earliest responses of barley epidermal cell defence against B. graminis attack and may be important in both the initiation and the development of effective papillae and cell death due to HR.