Elevations in cytosolic Ca²⁺ to chemical or hormonal transmitters and/or changes in physical parameters can control multiple cellular processes in endothelial cells and vasomotion in smooth muscle cells of the terminal branches of pre-capillary arterioles.The patterns, mechanism of initiation, and sources of Ca²⁺ involved in control of Ca²⁺ signalling induced by these factors in pre-capillary vessels are poorly understood. We have therefore investigated Ca²⁺ signalling 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 which were strongly potentiated by carbachol (50nM). Time to peak, half-time of relaxation, duration at 50% of peak in the presence of 50nM carbachol were increased from 0.19±0.02 to 0.48±0.04s, 0.29±0.02.19 to 0.59±0.03s, 0.17±0.02 to 0.55±0.06s (n=18), respectively and the frequency of Ca²⁺ puffs in the presence of carbachol was also significantly increased from 0.40±0.02 to 1.02±0.08Hz (n=18). Remarkably, carbachol-induced Ca²⁺ oscillations in endothelial cells could be observed after long (40-60min) exposures to Ca²⁺-free solutions (n=5) but were reversibly blocked by cyclopiazonic acid (SERCA blocker, n=9), 2-APB (IP₃Rs antagonist, n=5) or U73122 (phospholipase C inhibitor, n=6). In the same preparations and conditions, phenylephrine (10µM) and endothelin-1 (1-10nM) produced asynchronous Ca²⁺ waves in vascular myocytes which were independent of extracellular Ca²⁺ and could be observed after long (60-90 min) exposures to Ca²⁺-free solutions containing 2mM EGTA (n=9), little affected by ryanodine (n=11), but blocked by 2-APB (n=7) and U73122 (n=6). This was in marked contrast to rat mesenteric artery which under identical experimental conditions showed elevations of cytosolic Ca²⁺ in response to agonists which required both Ca²⁺ release and Ca²⁺ entry and cooperation of both RyRs and IP₃Rs channels.Carbachol activation of the endothelium had no effect on agonist-induced vasomotion in the pre-capillary arterioles but inhibited Ca²⁺ entry and vasoconstriction in rat mesenteric arteries. Inhibition of gap junctions by 18-β-glycyrrhetinic acid (50µM) inhibited communication between endothelial cells and myocytes in mesenteric arteries but had no effect in the pre-capillary arterioles. We suggest that, unlike larger diameter arteries, in pre-capillary arterioles, vasomotion evoked by agonists is insensitive to the endothelium and produced exclusively by intracellular Ca²⁺ waves in the form of asynchronous Ca²⁺ oscillations, mediated by IP₃Rs.