1. The roles of the endothelium-derived nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) in mediating vasodilator responses to acetylcholine and bradykinin were assessed in the ciliary vascular bed of the bovine isolated perfused eye preparation. 2. Vasodilatation to acetylcholine or bradykinin was unaffected by the nitric oxide synthase inhibitor, L-NAME (100 microM), or the cyclo-oxygenase inhibitor, flurbiprofen (30 microM), but was virtually abolished following treatment with a high concentration of KCl (30 mM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min). 3. Acetylcholine-induced vasodilatation was unaffected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels (K(+)(ATP)), but was significantly attenuated by TEA (10 mM), a non-selective inhibitor of K(+) channels. 4. The small conductance calcium-sensitive K(+) channel (SK(+)(Ca)) inhibitor, apamin (100 nM), and the large conductance calcium-sensitive K(+) channel (BK(+)(Ca)) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine-induced vasodilatation. In contrast, the intermediate (IK(+)(Ca))/large conductance calcium-sensitive K(+) channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response. 5. The combination of apamin (100 nM) with a sub-threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine-induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect. 6. In conclusion, blockade by a high concentration of KCl, by charybdotoxin, or by the combination of apamin with a sub-threshold concentration of charybdotoxin, strongly suggests that vasodilatation in the bovine isolated perfused eye is mediated by an EDHF.