Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall components involved in plant defense response to pathogen attack. In the present study, a resistant pearl millet (Pennisetum glaucum) cultivar, IP18292, was compared with a susceptible cultivar, 7042S, to investigate the contribution of HRGPs in the successful defense against the phytopathogenic oomycete S. graminicola. Northern hybridization using MeHRGP cDNA, a heterologous probe from cassava, indicated steady accumulation of HRGP transcripts, from 2 h.p.i. onwards with a maximum at 6 h.p.i., in the resistant cultivar. This is followed by HRGPs accumulation at about 8 h.p.i. as revealed by Western-blot analysis. Immunocytochemical localization by tissue printing and confocal immunofluorescence microscopy indicated cell walls of parenchymatic cells and the vascular tissue of coleoptile as sites of HRGP deposition. In vitro studies in the presence of horseradish peroxidase and H2O2 showed cross-linking of pearl millet HRGPs, which occurred parallel to isodityrosine accumulation. Inducible high isodityrosine content was also observed in vivo in the resistant cultivar. Here, H2O2 was found to accumulate as twin burst at 1 and 6 h.p.i., whereas in the susceptible cultivar only an early single peak was detectable. Moreover, the amount of hydroxyproline in HRGPs was about twice as high in the resistant as in the susceptible cultivar. These results suggest that cell wall strengthening in S. graminicola-infected resistant pearl millet is brought about by a combination of polypeptide cross-linking of isodityrosine as well as by the high content of hydroxyproline in HRGPs, and H2O2, in contrast to the susceptible plant.