Erwinia amylovora and Pseudomonas syringae pv. syringae produce elicitors of the hypersensitive reaction (HR), harphiEa and harpinPss, respectively. HarpinEa causes K+ efflux and extracellular alkalinization in suspension-cultured cells of tobacco. These responses are associated with disease resistance. We treated living, fixed, and permeabilized cells and protoplasts from tobacco suspension cultures with harpinPss, antiharpinPss antibody, and fluorochrome-tagged antibody and examined them with confocal laser microscopy. The fluorescent signal was localized in the outer part of the cell and was not observed in protoplasts. EGTA, a chelating agent that extracts Ca++ and pectins from cell walls, blocked harpinPss binding, as evidenced by the absence of fluorescent signal. The pH of the external medium of suspension cultures alkalinized in response to harpinPss and P. s. syringae. Bacteria and harpinPss-induced alkalinization of the extracellular medium were also completely blocked upon EGTA treatment Protoplasts alkalinized the medium at much reduced levels in response to P. syringae pv. syringae and did not alkalinize the medium in response to harpinPss. These results suggest that the cell wall is crucial for HR induction in the suspension culture cell.