beta(1,3)-Glucanases are a class of hormonally and developmentally regulated plant hydrolytic enzymes, which are also induced upon pathogen infection, suggesting a role of these hydrolases in the defense response to pathogens. We have purified several beta(1,3)-glucanases present in tobacco leaves from control plants and from plants treated with salicylic acid or infected with Pseudomonas syringae and studied in detail the subcellular localization of the different isoforms. Partial protein sequence analysis demonstrated that each of the different isoforms had a unique amino acid sequence and was therefore encoded by a different gene. We have also demonstrated that two of these isoforms, similar to the cytokinin/auxin-regulated isoforms previously isolated from tobacco cell suspensions, are located in the central vacuole. Upon salicylic acid treatment or P. syringae infection, three secreted isoforms are induced, belonging to the so-called pathogenesis-related proteins. These pathogenesis-related beta(1,3)-glucanases are all distinct from each other and also different from the vacuolar isoforms. We demonstrate that the vacuolar isoforms are not secreted to the extracellular spaces of the plant following pathogen infection, suggesting that any function they play in the plant defense response is restricted to an intracellularly coordinated defense process.