We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.