Acetylcholine (ACh) released at the neuromuscular junction (NMJ) triggers muscle fiber contraction. We tested whether this source of ACh also triggers vasodilation. Arterioles [diameter: 4th order (4A), 18 +/- 3 microns; 2nd order (2A), 35 +/- 2 microns] and feed arteries (60 +/- 4 microns) were observed in retractor muscle of anesthetized hamsters. During stimulation [25% duty cycle (500-ms train, 1 per 2 s) at approximately 40% of maximum isometric tension], a nicotinic receptor antagonist (tubocurarine, 10 microM) prevented contraction, yet 2A and 4A arterioles and feed arteries rapidly (< or = 5 s) dilated (by 9 +/- 2, 11 +/- 3, and 8 +/- 1 microns, respectively; P < 0.05); neither cholinergic innervation of the vasculature nor ACh release from endothelium was apparent. Vasodilator responses doubled (P < 0.05) with cholinesterase inhibition (eserine, 1 microM) and were abolished with muscarinic receptor antagonism (atropine, 10 microM). Microiontophoresis of ACh onto arterioles triggered vasodilation that conducted into feed arteries, confirming functional continuity between intramuscular and extraparenchymal resistance vessels. To determine whether ACh served as a vasodilator during exercise, vascular responses to muscle contraction were measured in the presence or absence of atropine. With 2.5% duty cycle (50 ms, 1 per 2 s), atropine attenuated vasodilation by 35% in 2A and 51% in 4A arterioles and by 65% in feed arteries. With 25% duty cycle, arteriolar dilation was unaffected by atropine, yet feed artery dilation was attenuated by 60%; this was accompanied by a 50% reduction in functional hyperemia. Our findings indicate that ACh "spillover" from NMJs can coactivate muscarinic receptors, giving rise to a dilation that is conducted into feed arteries. This ascending vasodilation is integral to the full expression of functional hyperemia.