We measured O2-, H2O2, .OH and leukotoxin biosynthesis in neutrophil plasma membrane. O2- was produced by respiratory burst oxidase in the membrane coupling with NADPH oxidation. Leukotoxin was biosynthesized in the system containing linoleate. Addition of SOD into the system doubled the amount of leukotoxin synthesized. Further addition of catalase into the system decreased leukotoxin formation. .OH and leukotoxin formation in the system was substantially increased by the presence of cytochrome c. In addition, leukotoxin was detected in the non-biological reaction mixture containing H2O2 and linoleate in the presence of heme iron. In this mixture, .OH and leukotoxin formation also showed a good correlation. From these results, it is evident that, in neutrophil cell membrane, leukotoxin is synthesized by .OH with linoleate.