We evaluated a method for the assessment of the phagocytic and bactericidal activity of human peripheral neutrophils against Staphylococcus aureus Cowan I, which is a modified version of the acridine orange staining technique originally described by Smith and Rommel (1977). The modification consisted of the use of free leukocyte suspensions rather than coverglass adhered leukocytes in order to avoid two main problems: the inefficient neutrophil adherence to glass that can be observed in specimens from patients with certain functional phagocyte defects, and the risk of selecting among neutrophils. An additional advantage of the modified procedure is that it permits a uniform bacteria: phagocyte ratio in different cell samples. The method was tested on 25 healthy adults and on four children with functional phagocytic defects (chronic granulomatous disease of infancy, Chediak-Higashi syndrome, and Rothmund-Thomson syndrome associated to persistent neutropenia and low chemotactic response). The neutrophils of all four patients showed a low bactericidal activity, with percent values of intracellular killed bacteria below the mean +/- 2 SD range observed in the healthy population at all incubation times tested (5, 15 and 30 min). A significant reduction in phagocytosis index and in % killed unopsonized S. aureus was observed in relation to bacteria treated with a pool of normal human serum. These results demonstrate the high sensitivity of the method, which could be used to determine intrinsic and extrinsic functional alterations in human neutrophils.