Endothelin is an endogenous vasoconstrictor likely to modulate the tone of retinal arterioles. We have previously demonstrated that this agonist can induce cyclical, inwardly directed Ca²⁺-activated Cl^– currents in ocular blood vessels (Curtis & Scholfield, 2000). In this study the effects of endothelin on Ca²⁺ signalling in arteriolar smooth muscle were examined at the cellular level using high speed Ca²⁺ imaging. Arterioles removed post mortem from Sprague Dawley rats (200-300g) were mechanically dispersed from fresh retinae using a fire-polished Pasteur pipette and microvascular smooth muscle (MVSM) cells loaded with 10µM Fluo-4AM for 2 hours. Changes in [Ca²⁺]_i were imaged in MVSM cell arrays (9-15 cells) using confocal scanning laser microscopy in line scan mode (500 scans/s). The scanline was oriented parallel with the vessel lumen, so that [Ca²⁺]_i was imaged simultaneously across the width of adjacent cells. Raw fluorescence data were extracted for graphical presentation using Image J (NIH), and [Ca²⁺]_i events were detected and analysed using custom-designed software. Although high concentrations of endothelin-1 produced synchronised increases in global [Ca²⁺]_i in adjacent MVSM cells, lower concentrations (10nM) induced rhythmical [Ca²⁺]_i oscillations (Fig. 1). These were often very regular in frequency within a given cell. The effects of endothelin were blocked by pre-exposure to BQ123 (100nM), an inhibitor of endothelin A-type receptors (n=5). Endothelin-1-induced [Ca²⁺]_i oscillations could also be reversed by subsequent application of tetracaine (100µM, n=6). They were also inhibited by the inositol 1,4,5-trisphosphate (IP₃) receptor blockers xestospongin C (10µM, n=7; and 50µM, n=8) and 2-aminoethoxydiphenyl borate (100µM, n=5). We conclude that endothelin-1, a physiological agonist of retinal MVSM cells, stimulates rhythmical Ca²⁺ oscillations in these microvessels via activation of endothelin A receptors. The response seems to be dependent on release of Ca²⁺ stores from the sarcoplasmic reticulum via both ryanodine receptors and IP₃ receptors, since inhibition of either pathway reversed the effects seen. This endothelin-induced activity may explain the Ca²⁺-activated Cl^– currents previously observed in the presence of this agonist (Curtis & Scholfield, 2000).