Using a recently developed in vitro preparation of vagal afferent pathways, we examined the role of TRPV1 and ASIC3 on the mechano- and chemosensitive properties of gastroesophageal sensory neurons. Esophagus, stomach, and the intact vagus nerves up to the central terminations were carefully dissected from TRPV1 and ASIC3 knockout mice and wild-type controls. The organ preparation was placed in a superfusion chamber to obtain intracellular recordings from the soma of nodose neurons during luminal stimulation of esophagus and stomach. The proximal esophagus and distal stomach were separately intubated to allow perfusion and graded luminal distension. In wild-type mice, mechanosensitive neurons were activated by low distension pressures and encoded stimulus intensity over the entire range tested. Luminal acidification significantly transiently increased the resting frequency but did not alter responses to subsequent mechanical stimulation. ASIC3 and TRPV1 knockout significantly blunted responses to distension compared with wild-type controls, with deletion of TRPV1 having a more significant effect than ASIC3 deletion. Luminal acidification did not activate mechanosensory neurons in ASIC3 and TRPV1 knockout mice. Our data demonstrate a role of TRPV1 in chemo- and mechanosensation of gastroesophageal afferents. ASIC3 may contribute to acid sensation but plays a more subtle role in responses to distending stimuli. Considering the importance of acid in dyspeptic symptoms and gastroesophageal reflux, TRPV1 or ASIC3 may be an attractive target for treatment strategies in patients who do not respond to acid suppressive therapy.