—We studied the mechanisms of retinal and choroidal vasorelaxation elicited by nitric oxide (NO) using piglet eyes. The NO donors sodium nitroprusside (SNP) and diethylamine-NONOate caused comparable concentration–dependent relaxation that was partially (≈40%) attenuated by the guanylate cyclase inhibitors methylene blue and LY83583 and reduced to a lesser extent (≈25%) by the inhibitor of cGMP–dependent kinase, KT 5823. In contrast, NO-induced dilatation (by NO donors and endogenous NO after stimulation with bradykinin) was substantially (≈70%) diminished by the K_Ca channel blockers tetraethylammonium (TEA), charybdotoxin, and iberiotoxin; by the cyclooxygenase inhibitors indomethacin and ibuprofen; by the prostaglandin I (PGI₂) synthase inhibitor trans-2-phenyl cyclopropylamine (TPC); and by the removal of endothelium; whereas relaxation of endothelium-denuded vasculature to SNP was unaltered by indomethacin, TPC, and charybdotoxin but was nearly nullified by methylene blue and the K_v channel blocker 4-aminopyridine. NO donors significantly increased PGI₂ synthesis and the putative PGI₂ receptor–coupled second messenger cAMP, from ocular vasculature (retinal microvessels and choroidal perfusate), and this increase in PGI₂ formation was markedly reduced by TPC, tetraethylammonium, charybdotoxin, and/or the removal of endothelium, but it was only slightly reduced by methylene blue and LY83583. Also, SNP and K_Ca channel openers NS1619 and NS004 caused an increase in PGI₂ synthesis in cultured endothelial cells, which was virtually abolished by K_Ca blockers. Finally, vasorelaxation to a cGMP analogue, 8-bromo cGMP, and protein kinase G stimulant β-phenyl-1,N²-etheno-8-bromoguanosine 3′:5′-cyclic monophosphate was mostly K_v dependent and, in contrast to NO, largely unrelated to PGI₂ formation. In conclusion, data indicate that NO-induced ocular vasorelaxation is partly mediated by cGMP through its action on smooth muscle, and more importantly, by stimulating PGI₂ formation of endothelial origin via a mechanism mostly independent of guanylate cyclase, which involves the opening of a K_Ca channel.