A procedure has been developed for the isolation of cell walls from the hyphae of the causal agent for barley leaf scald, Rhynchosporium secalis (Oudem) J.J. Davis. Based primarily on monosaccharide linkage analysis, but also on the limited use of linkage-specific glucan hydrolases and solvent fractionation, the walls consist predominantly of (1,3/1,6)-β-d-glucans, (1,3;1,4)-β-d-glucans, galactomannans of (1,2;1,6)-Manp residues and (1,5)-galactofuranosyl [(1,5)-Galf] side chains, rhamnomannans of (1,6)-Manp residues and rhamnopyranosyl [(1,2)-Rhap] side chains, and chitin; the walls also contain ≈23% (w/w) protein. Electron microscopy shows the presence of distinct inner and outer wall layers. Treatment of wall preparations with guanidine hydrochloride dissolves the outer layer and enables separate analysis of the inner and outer walls. The insoluble, inner wall layer is composed of (1,3/1,6)-β-d-glucans, galacto- and rhamnomannans, (1,3;1,4)-β-d-glucans and chitin, whereas the soluble outer wall material contains a high proportion of rhamnomannan, and smaller proportions of galactomannan, (1,3;1,4)-β-d-glucan and (1,3/1,6)-β-d-glucan with only trace levels of chitin. It was confirmed by immunochemical and enzymatic analysis that at least a portion of the (1,3;1,4)-β-d-glucan component of the inner wall exists as a (1,3;1,4)-β-d-glucan. The analyses not only provide information that is important for a complete understanding of the interactions between R. secalis and barley, but they also identify potential targets for the development of fungicides or resistant transgenic barley varieties.