Na(+)-H+ exchange activities were studied in vascular smooth muscle cells (VSMC) obtained from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The cytoplasmic pH of VSMC was detected by 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF), which was used as a fluorescent pH probe with a fluorescent microplate reader; this enabled us to measure the cytoplasmic pH of cells attached to multiwell culture plates. We measured cytoplasmic pH recovery in quiescent VSMC after acid loading with 20 mmol/l NH4Cl in HCO3(-)-free buffer. This recovery was inhibited either by removal of extracellular sodium or by addition of 5-N-ethyl-N-isopropyl amiloride (EIPA). The initial recovery rate was dependent on extracellular sodium concentration. Therefore, this change in cytoplasmic pH was presumed to be due to amiloride-sensitive Na(+)-H+ exchange. The Na(+)-H+ exchange activity represented by the cytoplasmic pH recovery rate was significantly higher in VSMC from SHRSP than in WKY cells at extracellular sodium concentrations greater than or equal to 20 mmol/l. In contrast, the steady-state cytoplasmic pH of quiescent VSMC from SHRSP was lower than that of WKY cells in HCO3(-)-free buffer. These findings seemed to contradict each other, and suggested that cytoplasmic pH was regulated not only by Na(+)-H+ exchange but also by other complicated mechanisms.