Spinal afferent neurons, with endings in the intestinal mesenteries have been shown to respond to changes in vascular perfusion. The mechanisms underlying this sensitivity were investigated in an in vitro preparation of mesenteric fan devoid of connections with the gut wall. Afferent discharge (AD) increased when vascular perfusion was stopped ("flow-off"), a response localized to the terminal vessels just prior to where they entered the gut wall. The "flow-off" response was compared following pharmacological manipulations designed to determine direct mechanical activation from indirect mechanisms via the vascular endothelium or muscle. In calcium-free conditions responses to "flow-off" were significantly augmented. In contrast, the MLCK inhibitor wortmannin (1 µM, 20min), did not affect the "flow-off" response despite blocking the vasoconstriction evoked by 10 µM L-phenylephrine. This ruled out active tension, generated by vascular smooth muscle, in the response to "flow off". Passive changes caused by vessel collapse during "flow-off" were speculated to affect sensory nerve terminals directly. The "flow-off" response was not affected by the N,P and Q-type Ca2+ channel blocker, -conotoxin MVIIC (1µM, i.a.) or the P2X-receptor/ion channel blocker, PPADS (50 µM). However, Ruthenium Red (50 µM), a blocker of non-selective cation channels greatly reduced the "flow-off" response and also abolished the vasodilator response to capsaicin . Our data support the concept that mesenteric afferents sense changes in vascular flow during "flow-off" through direct mechanisms, possibly involving non-selective cation channels. Passive distortion in the fan, caused by changes in blood flow, may represent a natural stimulus for these afferents, in vivo.