The aminoglycosides are ineffective against intracellular Staphylococcus aureus, which resides within lysosomes, despite a strong extracellular bactericidal activity. Since they are slowly concentrated within lysosomes it was hypothesized that acidity within these cell compartments might impair antibiotic activity. The bactericidal activity of amikacin alone and combined with lysosomotropic alkalinizing agents (LAA), which can alkalinize acidic cell compartments, was evaluated. Before antibiotic challenge, some cells were preincubated with amikacin for 3 days to allow intracellular accumulation of drug. No intracellular killing activity was shown when non-preincubated cells were used. Conversely, with preincubated cells, S. aureus was killed within 4 h when LAA were incorporated into the incubation media but not with amikacin alone. Enhanced antimicrobial activity correlated with increase in lysosomal pH. Intracellular accumulation of amikacin was not changed by LAA. These results provide evidence that acidic pH within lysosomes impairs amikacin in killing S. aureus.