Amiloride combined with subinhibitory concentrations of tobramycin is synergistic against Pseudomonas cepacia in vitro. It is known that amiloride blocks transmembrane sodium (Na+) flux in eukaryotic cells, but the mechanism of action in bacteria is unknown. Amiloride-mediated Na+ gradient changes might affect transcellular tobramycin transport, resulting in synergy between these two drugs. To examine this hypothesis, we studied the effect of extracellular [Na+] on amiloride-tobramycin synergy with a reference strain of P. cepacia. Control growth in Mueller-Hinton broth (MHB) alone, MHB supplemented with 200 mmol/L NaCl, and MHB dialyzed against a Na+-free solution was determined by absorbance at 650 nm over 3 hours. Inhibition in the presence of amiloride, tobramycin, or a combination was expressed as percentage of control growth. Growth in the presence of tobramycin alone was significantly higher in MHB + NaCl than in MHB or dialyzed MHB (81% +/- 5%; vs 57% +/- 5%; 35% +/- 4%; mean +/- SEM, respectively, p = 0.003). Growth in the presence of amiloride alone was not significantly different in the three media (84% +/- 4%; 80% +/- 3%; 80% +/- 1%; respectively, p = 0.746). Percent control growth was significantly lower when the two drugs were combined, but results were not statistically different among the three media (3.5% +/- 3%; 0.2% +/- 0.8%; 4% +/- 1%; respectively, p = 0.604). We conclude that growth inhibition of P. cepacia in the presence of tobramycin is antagonized by increasing extracellular [Na+]. This antagonistic effect of [Na+] is reversed by amiloride. Changes in [Na+] do not appear to directly affect amiloride-tobramycin synergy in P. cepacia.