When neutrophil cytoplasts (granule-free vesicles of cytoplasm enclosed by plasmalemma) were exposed to the chemotactic peptide formylmethionyl-leucyl-phenylalanine, no luminol-dependent chemiluminescence was detected, despite a pronounced production of superoxide anions and hydrogen peroxide. Addition of purified myeloperoxidase (MPO) or human serum albumin (HSA) to the cytoplasts before the stimulus resulted in a chemiluminescence response. In contrast to the bimodal response obtained from normal PMNL, only a single peak of chemiluminescence was obtained from the cytoplasts responding to the peptide. The time-course of the response obtained in the presence of albumin was more prolonged than the response obtained in the presence of MPO. Furthermore, the involvement of different oxidative metabolites in the MPO and the HSA systems, respectively, was demonstrated by the accumulated chemiluminescence effect obtained when MPO, but not HSA, was introduced in the measuring system after FMLP addition. From these results it can be concluded that there are at least two different light-generating mechanisms in FMLP-induced luminol-dependent chemiluminescence of neutrophil cytoplasts. One of these is dependent on myeloperoxidase and possibly related to the myeloperoxidase-hydrogen peroxide reaction, whereas the other one is hydrogen peroxide independent.