The release of Ca²⁺ from the sarcoplasmic reticulum (SR) of smooth muscle in retinal arterioles is associated with muscle contraction and vascular constriction (Tumelty et al, 2007). There is evidence that arachidonic acid and its metabolites play a role in neurovascular coupling in the retinal microvasculature (Metea & Newman, 2006). We therefore decided to test the effects of arachidonic acid on Ca²⁺ signalling in rat retinal arteriolar smooth muscle. Sprague-Dawley rats (300-500g) were killed with CO₂, enucleated, and retinal arterioles were mechanically dispersed and loaded with Fluo-4-AM (10μM). Ca²⁺ events were imaged using line-scan confocal laser microscopy and local and global changes in smooth muscle [Ca²⁺] assessed using normalized fluorescence (F/F₀). Adjacent regions of the same vessel were imaged under control conditions and in the presence of arachidonic acid to limit photodamage. Data was collected from a minimum of 7 vessels isolated from at least 4 animals, and has been summarized as the mean±SEM. Application of arachidonic acid (10μM) decreased the fraction of smooth muscle cells displaying spontaneous Ca²⁺-oscillations, from 51±8% in control conditions to 21±6% after 5 minutes of arachidonic acid exposure (P<0.05, Wilcoxon signed-rank test). The average frequency of Ca²⁺-oscillations in active cells was also decreased, from a control value of 0.057±0.005 cell^-1 s^-1, to 0.038±0.006 cell^-1 s^-1 after 5 min exposure to arachidonic acid (P<0.05, Mann Whitney U-test). Control protocols, in which the same vessel was imaged at 2 different sites at an interval of 5 minutes but with no application of arachidonic acid, showed no reduction in the number of active myocytes (40±10% and 39±9% of cells generated oscillations at 0 and +5mins, respectively; NS). The oscillation frequency in active cells was also unaffected, with an average value of 0.039±0.003 cell^-1 s^-1 initially, and 0.044±0.005 cell^-1 s^-1 after 5 minutes (NS). Arachidonic acid had no effect on the amplitude or full duration at half maximum (FDHM) of Ca²⁺-oscillations (see Table 1). These data suggest that arachidonic acid inhibits the generation of global Ca²⁺ events in retinal arteriolar myocytes, which would be consistent with a vasodilatatory action in the retinal microcirculation.