Our recent study demonstrated that the responses of rat pulmonary sensory neurons to TRPV1 activators were enhanced by increasing temperature (AJP Lung 294:L563-571, 2008), but the role of the TPRV1 channel in this potentiating effect could not be definitively evaluated. In the present study, we used whole-cell perforated patch-clamp technique to compare the responses of isolated nodose/jugular sensory neurons to chemical activators and increasing temperature between wild type (WT) and TRPV1-null (TRPV1-/-) mice. Our results showed that, in voltage-clamp mode, the peak inward current evoked by hyperthermia was not different between WT and TRPV1-/- neurons; however, the inward current evoked by 2-aminoethoxydiphenyl borate (2-APB), a common activator of TRPV1-3 channels, was greatly potentiated by increasing temperature from 36 to 40.5°C in WT neurons (n = 9; P < 0.01), but was not affected by the same change in temperature in TRPV1-/- neurons (n = 9; P = 0.54). Similarly, the inward current evoked by acid (pH 5.5), an activator of both TRPV1 channel and the acid-sensing ion channel, was enhanced by increasing temperature (n = 7; P < 0.05) in WT neurons, and this potentiating effect was absent in TRPV1-/- neurons (n = 13; P = 0.11). These results demonstrated that deletion of the TRPV1 channel does not significantly alter the stimulatory effect of hyperthermia on nodose/jugular neurons, but eliminates the potentiating effect of increasing temperature on the responses of these neurons to non-selective TRPV1 channel activators. This study further suggests that a positive interaction between these chemical activators and increasing temperature at the TRPV1 channel is primarily responsible for the hyperthermia-induced sensitization of these neurons.