In order t o quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together t o allow phagocytosis t o occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate o f the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase t o remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence i n a luminometer. This new method o f enumerating intracellular bacteria was compared to the conventional method o f viable counts and found t o correlate ( r = 0.78). The bioluminescence assay developed was found t o be a relatively rapid alternative method t o the techniques currently used t o enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects o f antimicrobial agents on intracellular pathogens.