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Previous investigators have demonstrated that polymorphonuclear neutrophils exhibit intense chemiluminescence (CL) during phagocytosis and the CL response can be used to study the cellular and humoral aspects of the phagocytic process. A microtechnique that used 10 microliters of whole blood as a source of phagocytes was developed and used to measure the phagocytic CL response during pregnancy. Increased sensitivity was achieved by the use of a high concentration of luminol (0.5 mM), prepared by sonication, as a CL amplifier. The high intensity of CL produced with luminol permitted the use of a scintillation counter in the IN coincidence mode, avoiding the necessity of dark-adapting the counting vials and reagents and working under subdued red light. The CL response was dose dependent on the number of phagocytes and/or particles (polystyrene spherules, opsonized zymosan, and E coli). The CL response was decreased by inhibitors that prevent particle uptake (iodoacetate and fluoride), inhibitors that prevent free-radical production (sodium benzoate and superoxide dismutase), and by inhibitors that inactivate myeloperoxidase (cyanide and azide). Results suggested that the phagocytic CL response in our assay system was dependent on O2 activation followed by the activated O2 species reacting with myeloperoxidase and chloride. The new technique was used to demonstrate a progressive increase with gestation in the phagocytic CL response in pregnancy and a rapid decrease to normal values at 1 week postpartum.