An increase in ventricular pressure can alter cardiac excitation and contraction. Recent report has demonstrated that fluid pressure (FP) suppresses L-type $Ca^{2+}$ current with acceleration of the current inactivation in ventricular myocytes. Since the L-type $Ca^{2+}$ channels known to be regulated by intracellular pH ($pH_i$), this study was designed to explore whether pressurized fluid flow affects pHi in isolated rat ventricular myocytes. A flow of pressurized (~16 dyne/$cm^2$) fluid, identical to that bathing the myocytes, was applied onto single myocytes, and intracellular $H^+$ concentration was monitored using confocal $H^+$ imaging. FP significantly decreased $pH_i$ by $0.07{pm}0.01$ pH units (n=16, P<0.01). Intracellular acidosis enhances the activity of $Na^+-H^+$ exchanger (NHE). Therefore, we examined if the NHE activity is increased by FP using the NHE inhibitor, HOE642. Although HOE642 did not alter $pH_i$ in control conditions, it decreased $pH_i$ in cells pre-exposed to FP, suggesting enhancement of NHE activity by FP. In addition, FP-induced intracellular acidosis was larger in cells pre-treated with HOE642 than in cells under the control conditions. These results suggest that FP induces intracellular acidosis and that NHE may contribute to extrude $H^+$ during the FP-induced acidosis in rat ventricular myocytes.