Hydrogen sulfide (H
2
S), produced by the enzyme cystathionine-γ lyase (CSE), dilates arteries by hyperpolarizing and relaxing vascular smooth muscle cells (VSMC) and CSE knock-out causes hypertension and endothelial dysfunction showing the importance of this system. However, it is not clear if H
2
S-induced VSMC depolarization and relaxation is mediated by direct effects on VSMC or indirectly through actions on endothelial cells (EC). We reported previously that disrupting EC prevents H
2
S-induced vasodilation suggesting H
2
S might act directly on EC. Because inhibiting large-conductance Ca
2+
-activated K
+
(BK
Ca
) channels also inhibits H
2
S-induced dilation, we hypothesized that H
2
S activates EC BK
Ca
channels to hyperpolarize EC and increase EC Ca
2+
which stimulates release of a secondary hyperpolarizing factor. Small mesenteric arteries from male Sprague-Dawley rats were used for all experiments. We found that EC disruption prevented H
2
S-induced VSMC membrane potential (
E
m
) hyperpolarization. Blocking EC BK
Ca
channels with luminal application of the BK
Ca
inhibitor, iberiotoxin (IbTx, 100 nM), also prevented NaHS-induced dilation and VSMC hyperpolarization but did not affect resting VSMC
E
m
showing EC specific actions. Sharp electrode recordings in arteries cut open to expose EC demonstrated H
2
S-induced hyperpolarization of EC while Ca
2+
imaging studies in fluor-4 loaded EC showed that H
2
S increases EC Ca
2+
event frequency. Thus H
2
S can act directly on EC. Inhibiting the EC enzyme cytochrome P
450
2C (Cyp2C) with sulfaphenazole also prevented VSMC depolarization and vasodilation. Finally, inhibiting TRPV4 channels to block the target of the Cyp2C product 11,12-EET inhibited NaHS-induced dilation. Combined with our previous report that CSE inhibition decreases BK
Ca
currents in EC, these results suggest that H
2
S stimulates EC BK
Ca
channels and activates Cyp2C upstream of VSMC hyperpolarization and vasodilation.
Use of hazardous event frequency to evaluate safety integrity level of subsea blowout preventer