Small arteries and arterioles exhibit vasomotion that can be synchronous over considerable distances. Vasomotion may assist perfusion during periods of altered metabolism or pressure. The mechanism of initiation, sources of Ca2+ and role of coupling between cells in vasomotion are poorly understood. We have therefore investigated this in in situ rat pre-capillary arterioles, using Fluo-4 and wide-field, confocal imaging to monitor changes in [Ca2+]i. Endothelial cells showed spontaneous Ca2+ events, ranging from Ca2+ puffs to Ca2+ waves. These events were resistant to 0-Ca2+ solutions, nifedipine and ryanodine, and potentiated by carbachol. Both the spontaneous and carbachol-induced Ca2+ events were reversibly blocked by cyclopiazonic acid (SERCA blocker), 2-APB (IP3Rs antagonist) or U73122 (phospholipase C inhibitor). In the same preparations and conditions, myocytes at rest generated Ca2+ sparks, which were blocked by ryanodine and augmented by caffeine (0.5–1mM). Phenylephrine (10μM) and endothelin-1 (1–20nM) produced asynchronous Ca2+ waves, which were associated with vasomotion, little affected by ryanodine, but blocked by 2-APB and U73122. Carbachol activation of the endothelium had no effect on agonist-induced vasomotion in the pre-capillary arterioles but inhibited vasomotion in rat mesenteric arteries. Inhibition of gap junctions by 18-β-glcerrhinic acid (50μM) inhibited communication between endothelial cells and myocytes in mesenteric arteries but had no effect on vasomotion evoked by agonists in the pre-capillary arterioles. We suggest that, unlike large diameter arteries, in pre-capillary arterioles, vasomotion evoked by agonists is insensitive to the endothelium and produced by intracellular Ca2+ waves in the form of asynchronous Ca2+ oscillations, mediated by IP3Rs.