There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.