Polymorphonuclear leukocytes derived from human peripheral blood, rabbit peritoneal exudates, and guinea pig peritoneal exudates generate measurable quantities of superoxide anion when challenged with opsonized zymosan, but not when challenged with zymosan that has not been opsonized. The generation of superoxide is dependent upon the concentration of cells and closely parallels the stimulation of the hexose mono phosphate shunt in the phagocytes. In contrast, no superoxide can be detected when rabbit alveolar macrophages (either normal or activated by prior vaccination of the animal with bacille Calmette-Guérin) or rabbit peritoneal macrophages are similarly treated. These data suggest that although superoxide anion may be involved in the bactericidal activity of the polymorphonuclear leukocyte, it is less likely to playa significant role in the bactericidal activity of the macrophage.