The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H202, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H202 production. Resting PMN exhibited slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reducing system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating, capacity within the phagocytic vacuole.Phagocytosis by polymorphonuclear leukocytes (PMN) ~ is accompanied by specific alterations in Abbreviations used in this paper: AT, 3-amino-I, 2, 4-triazole; Hanks' BSS, Hanks' balanced salt solution; LPS, lipopolysaccharide; NADH, ~/-nicotinamide adenine dinucleotide phosphate, reduced form; NBT, nitroblue tetrazolium; 02 , superoxide; PCMBS, the oxidative metabolism of the cell. Ingestion of particles or other stimulation of the surface triggers increases in oxygen consumption, hydrogen peroxide production, and hexose monophosphate p-chloromercuribenzene sulfonic acid; PMN, polymorphonuclear leukocyte; PS, polystyrene; SOD, superoxide dismutase; TNBT, tetranitrobiue tetrazolium.