The subcellular distribution of G protein subunits in the neutrophil was examined. Cells were nitrogen cavitated and subcellular organelles fractionated on discontinuous sucrose gradients. The presence of GTP-binding regulatory protein (G protein) alpha and beta/gamma subunits in each organelle was determined using three methods of analysis: specific binding of guanine nucleotide, ADP ribosylation by pertussis toxin, and immunoblot analysis with subunit-specific G protein antibodies. Both plasma membrane and cytosolic G protein components were detected. In contrast, neither the specific nor the azurophilic granules contained detectable G protein. Based on the ability of exogenous G protein beta/gamma subunits to increase the ADP ribosylation of the cytosolic form of G protein and upon the hydrodynamic behavior of the cytosolic protein, it is likely that this represents an uncomplexed G protein alpha subunit. Proteolytic mapping with Staphylococcus aureus V8 protease suggests the soluble alpha subunit is from Gn, the major pertussis toxin substrate of human neutrophils. Using quantitative analysis, the levels of the 40-kD G protein alpha subunit and of the 35/36-kD beta subunit in the neutrophil membrane were determined.