A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at -70 degrees C.