A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses. Meningococcal outer membrane structures (OMV) were adsorbed to fluorescent polystyrene beads, opsonized with serum, and exposed to leukocytes. FCM parameters of phagocytosis were evaluated in combinations with oxidative burst indicators. Rhodamine-123 was the most sensitive indicator and was compatible with quantitation of phagocytosis. The phagocytosis and oxidative burst responses induced by OMV beads were dependent on both antigens and opsonins. Increased human opsonic responses against OMV were induced during clinical meningococcal disease. A dissociation was noted between phagocytosis and oxidative burst in individual cells, indicating that functional opsonins against OMV components may differ in their ability to stimulate phagocytosis and oxidative burst responses. The method facilitates evaluation of purified bacterial structures as mediators of opsonin-dependent phagocytosis and intracellular oxidative microbicidal mechanisms, which is of interest in the complex process of selecting bacterial antigens as constituents of certain vaccines.