The high antibacterial activity and selectivity of aminoglycosides and their low activity against intracellular bacteria associated with eukaryotic cells make them the antibiotics of choice for the elimination of extracellular bacteria during intracellular studies. Given the evidence that aminoglycosides can penetrate the eukaryotic cell membrane, the goal of this study was to examine the influence of aminoglycosides on macrophage-associated Salmonella. Herein, we show that gentamicin, kanamycin, and tobramycin at concentrations between 15 to 150 microg ml(-1) do not kill intracellular Salmonella but have other effects on the bacterial physiology. By using Salmonella enterica serovars Typhimurium and Virchow harboring luciferase reporter plasmid, we observed that the light produced by intracellular Salmonella declined immediately upon exposure to aminoglycosides, indicating that the bacteria were under stress. The extent of this effect was dependent on the macrophage host, on the identity of the aminoglycoside and its concentration, on the exposure time, and on the Salmonella serovar. Salmonella associated with Nramp1-negative macrophages, in which the phagosomal pH is higher, were more susceptible to aminoglycosides than Salmonella associated with Nramp1-expressing macrophages. These results verify that aminoglycosides affect intracellular bacteria and that the extent of this effect is dependent on the acidity level within the phagosome, suggesting that for the study of intracellular bacteria, the aminoglycoside concentration should be limited to two to five times the MIC for the bacterial strain studied. This precaution should guarantee the complete execution of extracellular bacteria with minimal effects on the intracellular bacteria and the host cells.