Little is known about Ca²⁺ signalling in microvessels such as pre-capillary arterioles and sphincters. We have therefore investigated the role of ryanodine and IP₃ receptors (RyR and IP₃R) in three different vessels (pre-capillary sphincters, pre-capillary arterioles and mesenteric resistance arteries) in response to stimulation by phenylephrine (PhE), endothelin-1 (ET-1) and caffeine. A minimum of 17 vessels from 15 humanely killed rats were studied by confocal microscopy. In small mesenteric arteries (mean diameter 230±21 µm) PhE (10 µM) or ET-1 (10nM) induced asynchronous Ca²⁺ waves followed by synchronous Ca²⁺ oscillations. In Ca²⁺-free solution, these oscillations were replaced by asynchronous Ca²⁺ waves. Caffeine (10 mM) induced large Ca²⁺ transients which were resistant to removal of extracellular Ca²⁺ for up to 40 min. Ryanodine (50 µM) abolished both PhE and caffeine-induced Ca²⁺ transients while the IP₃R inhibitor, 2-APB, inhibited PhE and ET-1 but not caffeine-induced Ca²⁺ transients. In contrast, in pre-capillary arterioles PhE and ET-1 produced only asynchronous Ca²⁺ oscillations which were resistant to removal of extracellular Ca²⁺. Caffeine induced a Ca²⁺ transient which was abolished by ryanodine. In these pre-capillary arterioles, the responses to PhE and ET-1 were abolished by 2-APB but not by ryanodine. Long exposures (~90 min) to Ca²⁺-free solution had no significant effects on agonist- or caffeine- induced SR Ca²⁺ release in pre-capillary myocytes. In sphincters, neither PhE nor caffeine had any effect on Ca²⁺ signalling but ET-1 produced oscillations which were independent of extracellular Ca²⁺ and blocked by 2-APB. The frequency of these Ca²⁺ oscillations was 1.6±0.2 times higher than in myocytes of pre-capillary arterioles. The data obtained show clear differences in the types of Ca²⁺ signals and the mechanisms generating them in the final branches of the arterial system. We propose the following; the small mesenteric arteries respond with Ca²⁺ waves to both circulating and local factors by Ca²⁺ releases through RyR and IP₃R channels. The Ca²⁺ waves lead to Ca²⁺ entry and produce synchronised Ca²⁺ oscillations. Pre-capillary arteriolar myocytes also are responsive to circulating and local factors. However, they do so only via IP₃R channels and Ca²⁺ entry mechanisms do not play a role. Finally at the level of pre-capillary sphincters, myocytes are responsive only to local factors via IP₃-mediated Ca²⁺ releases and not Ca²⁺ influx. These differences in Ca²⁺ signalling mechanisms will contribute to the functional differences between these blood vessels.