Treating NIH3T3 fibroblast cells with Pyrularia pubera thionin (100 μg/ml) stimulated release of labelled free fatty acids from phospholipids biosynthetically labelled by incorporation of [3H]arachidonic acid. Since Pyrularia thionin exhibited no detectable phospholipase activity, it was concluded that the release response represented activation of endogenous phospholipases in the cells. The phospholipase activated by Pyrularia thionin (100 μg/ml) stimulated the release of 16% of the incorporated [3H]arachidonate in the presence of 1.8 mM extracellular calcium with maximum activation at 90 min following an initial lag period of about 20 min. The release response exhibited little dependence on extracellular calcium at this thionin concentration, but at concentrations 20 μg/ml, extracellular calcium appeared to be inhibitory to phospholipase activation. Some characteristics of the fatty acid release response are consistent with activation of a lysosomal phospholipase being part of the cellular response to Pyrularia thionin. Activation of a lysosomal enzyme can occur independently or as a result of coordinate activation of the whole lysosome, which would expose other cellular components of degradative lysosomal enzymes. Consistent with coordinate activation of lysosomal enzymes, Pyrularia thionin also stimulates the production of small, trichloroacetic acid-soluble peptides and nucleic acid fragments from biosynthetically-labelled RNA and proteins in treated cells. It is not clear from the results obtained what role, if any, activation of lysosomal enzymes plays in the overall toxic response to Pyrularia thionin in NIH3T3 cells. However, Pyrularia pubera thionin may represent a useful tool for studying the regulation of lysosomal enzymes and their roles in cells.