Actively phagocytizing polymorphonuclear leukocytes (PMN) emit light or chemiluminescence (CL) which has been shown to be linked to the oxidative activity of the PMN. The measurement of CL has been demonstrated to be a useful tool for the in vitro assessment of intracellular and opsonophagocytic function of PMN. We have increased the sensitivity of the CL measurement by the addition of luminol to the in vitro reaction of PMN, bacteria, and serum. The presence of luminol, which can be oxidized to emit light, amplifies the detection of CL and PMN cellular activity. This amplification effectively reduces the number of PMN that are necessary for assessment of PMN function from 1 x 10(7) to as low as 2 x 10(4) PMN/assay and permits the evaluation of PMN function in severely neutropenic patients (100 PMN/mm3) in whom cellular PMN function has been heretofore extremely difficult to assess by other methodology. When this luminol-dependent CL method was used, three of eight neutropenic leukemic patients with gram-negative septicemia were found to have deficient opsonic activity and/or increased or depressed cellular oxidative activity. Because the initial slope of CL is dependent on the amount of serum and heat-labile factors, this method can also be used effectively as a simple technique for the analysis of specific rates of opsonophagocytosis of various microorganisms. Additionally, this method can detect the cellular PMN abnormalities of chronic granulomatous disease and myeloperoxidase deficiency. The luminol-dependent CL method is a simple, sensitive, reproducible technique that provides useful information about PMN metabolic activity, particularly in studies in which the number of PMN is limited.