Vasodilatory factors produced by the endothelium are critical mediators of blood pressure and flow. We hypothesised that endothelial signals are transduced to underlying smooth muscle by vanilloid transient receptor potential (TRPV) channels. Experiments were performed using cerebral arteries harvested from humanely killed male Sprague-Dawley rats. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells (n=3). In patch-clamp experiments employing freshly isolated cerebral myocytes, whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4α-phorbol 12,13-didecanoate (4α-PDD, 5 μM, n=3) and the endothelium-derived arachidonic acid metabolite 11, 12 epoxyeicosatrienoic acid (11,12 EET, 300 nM, n=6). High-speed laser scanning confocal microscopy was used to evaluate the effects of TRPV4 activation on intracellular Ca²⁺ dynamics in cerebral artery smooth muscle cells loaded with the fast Ca²⁺ indicator dye fluo-4. Values are means±S.E.M. 11,12 EET increased the frequency of unitary Ca²⁺ release events (Ca²⁺ sparks) via ryanodine receptors located on the sarcoplasmic reticulum (control, 0.18±0.02 Hz, n = 16; 11,12-EET, 0.29±0.03 Hz, n=13; P<0.05, t test). EETs-generated Ca²⁺ sparks activated nearby sarcolemmal large conductance Ca²⁺-sensitive K⁺ (BK_Ca) and increased the frequency of transient K⁺ currents (referred to as ‘spontaneous transient outward currents’ or ‘STOCs’) (control, 0.475±0.15 Hz, vehicle, 0.472±0.12 Hz, 11,12 EET (100 nM), 1.1±0.34 Hz*, n=9 for all groups *P<0.05 vs. all other groups, one-way repeated measure ANOVA). 11,12 EET-induced increases in Ca²⁺ spark and STOC frequency persisted when voltage-dependent Ca²⁺ channels (VDCCs) were inhibited (n=5 for both groups). Conversely, EETs-induced changes in Ca²⁺ spark and STOC frequency were blocked when external Ca²⁺ was reduced from 2 mM to 10 μM (n=5 for both groups), suggesting that these response require extracellular Ca²⁺ influx via channels other than VDCCs. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization (change in membrane potential in response to 300 nM 11,12 EET: sense, -10.3±0.18 mV, n=6 vs. antisense, -0.5±1.8 mV, P<0.05, two-way ANOVA) and vasodilatation (change in luminal diameter in response to 11,12 EET (100 nM): sense, 37.1±8.2 μm vs. antisense, -6.7±4.4 μm, n=5 for both groups, P<0.05, two-way repeated measures ANOVA). Thus, we conclude that TRPV4 forms a novel Ca²⁺ signalling complex with ryanodine receptors and BK_Ca channels that elicits smooth muscle hyperpolarization and arterial dilatation via Ca²⁺-induced Ca²⁺ release in response to an endothelial-derived factor.