A putative reservoir of functional plasma membrane proteins, the secretory vesicle identified by latent alkaline phosphatase and tetranectin, has previously been demonstrated based on indirect evidence (Borregaard, N., Miller, L. J., and Springer, T. A. (1987) Science 237, 1204-1206; Borregaard, N., Christensen, L., Bjerrum, O. W., Birgens, H. S., and Clemmesen, I. (1990) J. Clin. Invest. 85, 408-416). Difficulties in separating plasma membranes from this entity by density gradient centrifugation has prohibited discriminative dynamic and quantitative studies of secretory vesicles and plasma membranes. By combining density centrifugation with free flow electrophoresis we overcame this obstacle. Freshly prepared unperturbed human neutrophils were subjected to nitrogen cavitation followed by density centrifugation on Percoll gradients. Light membrane fractions containing plasma membranes and secretory vesicles were applied to high voltage free flow electrophoresis on an Elphor VaP 22. Plasma membrane vesicles, identified by HLA class I antigen mixed enzyme-linked immunosorbent assay (Bjerrum, O. W., and Borregaard, N. (1990) Scand. J. Immunol. 31, 305-313) and 125I applied to cells before cavitation, were clearly separated from secretory vesicles. Electron microscopy revealed a morphology typical of plasma membranes in the former fraction and a population of vesicles with markedly different appearance in the latter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles demonstrated distinct differences in protein patterns between the two fractions. Superoxide generating capacity induced by sodium dodecyl sulfate and cytosol, an entity traditionally ascribed to the plasma membrane, was largely confined to fractions containing secretory vesicles. Thus, the majority of membrane-bound NADPH oxidase components of light membranes of human neutrophils colocalize with secretory vesicles.