Complete protection of Staphylococcus aureus Wood 46 from gentamicin bactericidal activity was documented for microorganisms located within polymorphonuclear leukocytes. The highest, still ineffective gentamicin concentration tested in the phagocytic assay was 80 times higher than the minimal concentration required to kill uningested organisms. Extracellular gentamicin activity was unaffected by the phagocytic process as demonstrated by microbiological and enzymatic assays, and liberation of intracellular S. aureus by lysis of neutrophils showed the bacteria to be fully susceptible to the antibiotic. These results were corroborated by studies performed with [14C]gentamicin; binding of the labeled antibiotic by resting neutrophils, or by neutrophils ingesting live, killed S. aureus or endotoxin-coated paraffin particles, showed no statistical differences and never exceeded 20% of the extracellular concentration. These results show that intraleukocytic S. aureus are protected from the bactericidal action of gentamicin and suggest that this protection can be explained by poor intracellular penetration of the antibiotic.