Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pHo). Results from recentin vivostudies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pHo (6.0 < pH < 7.0) than predicted from thein vitroexperiments. A clear explanation for this discrepancy between thein vitroandin vivodata has not yet been provided. We report here that intracellular acidification induced by a moderately low pHo (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin-evoked TRPV1 current (ICAP) in a reversible manner in whole-cell patch-clamp mode and shifted the concentration-response curve to the right. Likewise, the concentration-response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressedICAP in inside-out patch mode. In cell-attached patch mode, the single-channel activity ofiCAP was significantly attenuated by intracellular acidosis that was induced by a decrease in pHo (6.4). These results suggested that intracellular acidification induced by a low pHo inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37°C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pHo (6.4) at a physiological temperature (37°C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pHo, and this phenomenon is due to attenuation of TRPV1 channel activity by low-pHo-induced intracellular acidification.