In the present study, we investigated the effect of three different sources of hydrogen sulfide (H2S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H2S-donating moiety; l-cysteine, a substrate for endogenous production of H2S and GYY 4137, a slow donor of H2S. We also examined the contribution of prostaglandins to the pharmacological actions of the H2S donors on release of [3H]-norepinephrine ([3H]NE) triggered by electrical field stimulation. ACS67, l-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [3H]NE release from isolated bovine iris-ciliary bodies without affecting basal [3H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and l-cysteine on stimulated [3H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-β-synthase and glibenclamide, a KATP channel blocker reversed the inhibition of evoked NE release induced by the H2S donors. We conclude that H2S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H2S and prostanoids, and is mediated by an action on KATP channels.