Abstract Race nonspecific resistance of barley against the barley powdery mildew fungus (Blumeria Graminis f.sp. Hordei, Speer, Bgh) is mediated by recessive mlo alleles and is controlled by at least two additional genes 'required for ml o-specified disease resistance' (Ror1 and Ror2). The pathogenesis-related accumulation of hydrogen peroxide (H(2)O(2)) was comparatively analysed in a susceptible barley line (Hordeum vulgare L. Cv Ingrid, genotype Mlo Ror1, Ror2), a resistant Ingrid backcross line carrying the mutant allele mlo5 (BCIngrid-mlo5, genotype mlo5 Ror1 Ror2), and in the moderately susceptible mutants A44 and A89 (genotypes mlo5 Ror1 ror2 and mlo5 ror1-2 Ror2, respectively). In situ localization of H(2)O(2) was performed by microscopic detection of 3,3-diaminobenzidine (DAB) polymerization. In BCIngrid-mlo5, penetration resistance against Bgh attack was closely correlated to H(2)O(2) accumulation in cytoplasmic aggregates and cell wall appositions beneath the appressorium. In contrast, H(2)O(2) accumulation was almost completely absent in susceptible Ingrid. Lines with mutations in Ror genes showed less H(2)O(2) accumulation beneath appressoria, but more interaction sites with whole cell H(2)O(2) accumulation and hypersensitive cell death response than resistant BCIngrid-mlo5. Thus, mutations in Ror1 or Ror2 genes influence the cellular pattern of H(2)O(2) accumulation in mlo plants attacked by Bgh. The data support the hypothesis that H(2)O(2) accumulation is involved in resistance to fungal penetration.