Carbon monoxide (CO) is endogenously produced by hemoxygenase (HO) and is involved in vascular, neural, and inflammatory responses in mammals. However, the biological activities of CO in non-mammalian vertebrates is unknown. To this extent, we utilized smooth muscle myography to investigate the effects of exogenously applied CO (delivered via a water-soluble CO-releasing molecule, CORM-3) on isolated lamprey (Petromyzon marinus) dorsal aortas and examined its mechanisms of action on trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM-3 dose-dependently relaxed all vessels examined. Trout EBA were 2-fold more sensitive to CORM-3 when precontracted with norepinephrine (NE) than KCl and CORM-3 relaxed 5-fold more of the NE- than KCl-induced tension. Glybenclamide (10 µM), an ATP-sensitive potassium channel inhibitor, inhibited NE-induced contraction, but did not affect CORM-3-induced relaxation. NS-2028 (10 µM), a soluble guanylyl cyclase inhibitor, had no effect on a NE-contraction, but inhibited a subsequent CORM-3-induced relaxation. Zinc protopophyrin-IX (ZnPP-IX, 0.3-30 µM), a HO inhibitor, elicited a small, yet dose-dependent and significant increase in baseline tension but did not have any effect on subsequent NE-induced contractions or a NO-induced relaxation (via sodium nitroprusside). [ZnPP-IX] greater than 3 µM, however, significantly reduced the predominant vasodilatory response of trout EBA to hydrogen sulfide (H₂S). These results implicate an active HO/CO pathway in trout vessels having impact on resting vessel tone and CO-induced vasoactivity that is at least partially mediated by soluble guanylyl cyclase.