P hagocytic vesicles from rabbit lung macrophages produced superoxide in the presence of NADH or NADPH. At 37°C, these vesicles generated 51±7.8 nmol O₂⁻/min per mg protein in the presence of 0.5 mM NADPH. The apparent K_m for NADPH and NADH (66 and 266 μM, respectively), the pH optimum for the reaction (6.9), and the cyanide insensitivity were similar to properties of plasma membrane-rich fractions of stimulated polymorphonuclear leukocytes studied by others. The activity of the phagocytic vesicles was trypsin sensitive. The specific superoxide-generating activity of macrophage phagocytic vesicles isolated from cells incubated up to 90 min with phagocytic particles remained constant.Calcium in micromolar concentrations inhibited the NADPH-dependent O₂⁻-generating activity of phagocytic vesicles. In a physiological ionic medium (100 mM KCl, 2.5 mM MgCl₂, 30 mM imidazole-HCl, pH 6.9), a maximal inhibition of O₂⁻ generation by phagocytic vesicles of 80% was observed at 40 μM free Ca²⁺. The half maximum inhibitory effect was at 0.7 μM Ca²⁺. Variations of the calcium concentration resulted in rapid and reversible alterations in O₂⁻-forming activity. Preincubation of phagocytic vesicles in the presence of EGTA rendered their O₂⁻ generation rate in the presence of NADPH insensitive to alterations in the free calcium concentration. This desensitization by low EGTA concentrations (≤100 μM) was reversible by the addition of excess calcium, but desensitization by high EGTA concentrations (>1 mM) was not reversible by the addition of calcium either in the presence or absence of purified rabbit lung macrophage or bovine brain calmodulins. Furthermore, trifluoperazine, a drug that inhibits calmodulin-stimulated reactions, did not alter the activity or the calcium sensitivity of the superoxide-generating system of sensitive phagocytic vesicles.Peripheral plasma membrane vesicles (podosomes) prepared by gentle sonication of macrophages possessed on O₂⁻-generating system with similar properties to those of phagocytic vesicles.We conclude that the activated O₂⁻-generating system of rabbit lung macrophages has its initial localization in the plasmalemma and undergoes subsequent internalization into phagocytic vesicles, where it can function for prolonged periods of time. Calcium at concentrations likely to exist in macrophage cytoplasm exerts a regulatory effect on the activated system.