Abstract—Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)- and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I–deficient (cGKI–/–) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI–/–arteries. However, contractions of cGKI–/–arteries and aortic rings were reduced by high concentrations (10 &mgr;mol/L) of 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca2+-activated K+(BKCa) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BKCachannels only in vascular smooth muscle cells isolated from wt cGKI+/+mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BKCa. NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI–/–vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK.