A modified method for measurement of intracellular killing of Staphylococcus aureus in human neutrophil granulocytes is described. After phagocytosis of S. aureus the extracellular bacteria were sufficiently removed by repeated centrifugations and washings of the granulocytes. The optimal conditions for incubation of granulocytes for measurement of intracellular killing were found to be 37 degrees C in the presence of 5% CO2. Under these conditions, stable pH, the viability and the capacity of the granulocytes for superoxide anion generation were preserved. The number of intracellular viable bacteria was determined after lysis of the granulocytes, which should be done in H2O at pH 11 to ensure sufficient cell lysis. The kinetics and individual variation of the intracellular killing are described. The intra- or extracellular location of surviving bacteria was studied. After approximately 8 h incubation we observed intracellular growth of S. aureus followed by lysis of granulocytes and extracellular growth of bacteria. Consequently, the incubation period should not be extended beyond 5 to 8 h when the assay is used to study the effects of antibiotics on intracellular killing.