Participation of reactive oxygen species (ROS) in the changes in macrophage membrane potential resulted from effects of different agonists has been studied. Treatment of macrophages with chemotactic peptide fMLP or platelet-activating factor (PAF) caused a brief depolarization followed by a long-lasting hyperpolarization. Lipopolysaccharide and interferon-gamma only depolarized the plasma membrane. Chemiluminescence measurements indicated that only fMLP and PAF activated macrophages to release ROS. The hyperpolarization response of the cell was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2.- -generating system, xanthine plus xanthine oxidase, caused a marked hyperpolarization. In all the cases, the hyperpolarization induced by fMLP, PAF and O2.- -generating system was found to depend on the concentration of intracellular Ca2+ and extracellular K+. Furthermore, in the presence of quinidine, a blocker of Ca2+-dependent K+ conductance fMLP and PAF caused only prolonged depolarization while the effect of O2.- was reduced to a minimum. These data suggest that the macrophage hyperpolarization response to fMLP and PAF involves superoxide-mediated Ca2+-dependent alteration of the relative membrane permeability to K+.