Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22-phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.