A very sensitive chemiluminescence (CL) method was developed for the determination of respiratory burst products generated by the NADPH-oxidase in human neutrophils. Despite the fact that the CL reaction is peroxidase dependent, hydrogen peroxide was found not to participate in the light generating reaction. Phagocytic cells were mixed with isoluminol, a chemiluminescence substrate that detects extracellularly released oxygen species only. Owing to the fact that the availability of released cellular myeloperoxidase is a limiting factor in the reaction, extra peroxidase (horseradish peroxidase; HRP) has to be added to the measuring system. From the fact that the response to phorbol myristate acetate (PMA), as well as to formyl-methionyl-leucyl-phenylalanine (fMLP) was almost totally inhibited by superoxide dismutase (SOD), we conclude that O2.- is the reacting oxygen species measured. The assay system described is well suited for real-time studies of superoxide anion release from activated neutrophils. With the technique, the release of O2.- can be detected from as few as 250 neutrophils.