Abstract: Medicago sativa was inoculated with Erysiphe pisi. The colorimetric method was used to study the timing and intensity of ^·O2- and H₂O₂ bursts in leaves, and histochemical methods were used to study localization of ^·O2- and H₂O₂ accumulation. In addition, paraffin sections were made from infected leaves to observe structural changes in those leaves. It was found that after inoculation, ^·O2- and H₂O₂ bursts in Qingyang alfalfa (resistant cultivar) were observed as two peaks, appearing 4 and 48 h after inoculation for ^·O2- and 4 and 24 h after inoculation for H₂O₂, respectively, with the intensity of the first peak higher than that of the second peak. By contrast, in Debao alfalfa (susceptible cultivar), no obvious fluctuation in ^·O2- content appeared and only the one peak for H₂O₂ accumulation was seen. Staining to reveal zones of localization of reactive oxygen species (ROS) showed that blue-stained ^·O2- deposition sites did not occur at the surface of or inside of leaves, while brown-stained H₂O₂ deposition sites were observed in the cell wall of upper and lower epidermal cells, and the cytoplasm of infected mesophyll cells. Most importantly, we report here for the first time that the tissue structure of alfalfa leaves was changed after infection with E.pisi. Palisade cells changed from a long-cylindrical cell type to groups of several subrotund cells, so that it was difficult to distinguish the palisade tissue and spongy tissue cells. Our results show that for the different plant species and pathogen resistance combinations investigated, the timing and intensity of ROS accumulation differed, as did the identity of the chemical species generated in activating the defense reaction. The structural and morphological change of mesophyll cells may be one component of an effective response against powdery mildew infection in M. sativa.