Recent studies suggest that the capsaicin receptor (TRPV1) may play a role in visceral mechanosensation. To address the potential role of TRPV1 in vagal sensory neurons, we developed a new in vitro technique allowing us to determine directly TRPV1 expression in physiologically characterized gastric sensory neurons. METHODS: Stomach, esophagus and the intact vagus nerve up to the central terminations were carefully dissected and placed in perfusion chamber. Intracellular recordings were made from the soma of nodose neurons during mechanical stimulation of the stomach. Physiologically characterized neurons were labeled iontophoretically with neurobiotin and processed for immunohistochemical experiments. RESULTS: Based on action potential responses triggered by stimulation of the upper thoracic vagus with a suction electrode, essentially all abdominal vagal afferents in mice conduct in the C fiber range. Mechanosensitive gastric afferents encode stimulus intensities over a wide range without apparent saturation when using punctate stimuli. Nine of 37 mechanosensitive vagal afferents expressed TRPV1 immunoreactivity, with 8 of the TRPV1 positive cells responding to stretch. A small number of mechanosensitive gastric vagal afferents express neurofilament heavy chains and did not respond to stretch. CONCLUSIONS: By maintaining the structural and functional integrity of vagal afferents up to the nodose ganglion, physiological and immunohistochemical properties of mechanosensory gastric sensory neurons can be studied in vitro. Using this novel technique, we identified TRPV1 immunoreactivity in only one fourth of gastric mechanosensitive neurons, arguing against a major role of this ion channel in sensation of mechanical stimuli under physiological conditions.