Viscerofugal neurons can modulate gastrointestinal motility through peripheral reflex pathways involving prevertebral ganglia. Indirect studies of viscerofugal neuron populations suggest they might be directly mechanosensitive. Previously, we showed that identified single viscerofugal neurons can be recorded extracellularly from mesenteric nerve trunks of in vitro preparations of guinea–pig intestine. Purpose: To study characteristics of viscerofugal neuron mechanosensory responses. Methods: Flat-sheet preparations of guinea-pig distal colon with or without circular muscle removed were studied in vitro. Mesenteric nerve trunks were dissected and recorded with extracellular electrodes. Single units, DMPP-sensitive, but capsaicin-insensitive, were considered viscerofugal neurons since we have shown that these criteria correlated statistically with the presence of viscerofugal neurons. Von Frey hairs (100-200mg) were used to probe focal DMPP-responsive sites. Circumferential length and tension was modulated pharmacologically under isotonic or isometric conditions. Results: Eighteen units were identified in preparations without circular muscle (N=6) by focal DMPP (1mM) application. All units responded promptly to von Frey hair probing in Ca²⁺-free Krebs. Thirteen units (N=6), identified in preparations with circular muscle, were stretched 1-4mm above resting length. All units increased firing rate upon circumferential distension in Ca²⁺- free Krebs. Under near-isometric conditions, the L-type Ca²⁺ channel agonist BAY K8644 (1μM) increased circumferential tension to 250% of basal tension (2.1±1.0g vs 6.1±2.5g, p<0.001, N=6) while viscerofugal neuron firing rate was unchanged (2.0±2.3 vs 2.0±2.0Hz, NS, paired t-test). Under near-isotonic conditions, BAY K8644 (10μM) increased contractility-associated changes in circumferential length (1.3±0.8mm vs 4.2±1.2mm, p<0.001, t-test, N=6). Viscerofugal neurons preferentially fired at greater circumferential lengths (17 units, 2-4g counter-loads, p<0.001, ANOVA). Conclusion: All viscerofugal neurons we identified were directly mechanosensitive which respond potently as length, not tension receptors