In type-I vestibular hair cells (VHCs), the mechanisms involved in intracellular calcium homeostasis have not yet been established. In order to investigate the involvement of an Na(+)-dependent ionic exchanger in the regulation of cytosolic free calcium concentration, we analyzed the effect of the removal of external sodium on the cytosolic concentration of calcium ions ([Ca2+]i), sodium ions ([Na+]i), and protons (pHi). These concentrations were measured in type-I VHCs isolated from guinea pig labyrinth, using Fura-2, sodium benzofuran isophtalate (SBFI), and 1,4 diacetoxy-2,3 dicyanobenzol (ADB) respectively. Complete replacement of Na+ in the superfusion solution with N-methyl-D-glucamine (NMDG+), reversibly increased [Ca2+]i by 276 +/- 89% (n = 46) and decreased [Na+]i by 23 +/- 6% (n = 14). Both responses were prevented by removing external Ca2+ or chelating internal Ca2+. This suggests the presence of coupled Ca2+ and Na+ transport. The [Ca2+]i increase evoked by Na(+)-free solution was reduced by about 55% with the application of amiloride derivatives and was totally abolished in the presence of high [Mg2+]o. No pHi variation was detected during [Na+]o reduction. In the absence of external K+, the Na(+)-free solution failed to induce [Ca2+]i increase; the readmission of external K+ restored the [Ca2+]i response. These results are consistent with a Na(+)-Ca2+ exchanger operating in reverse mode. An K+ dependence of this exchange is also suggested.