This study examined the response to nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors increased the activity of a 205-pS K⁺ channel recorded from vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade of guanylyl cyclase activity with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10⁻⁵ M) did not alter the effect of NO on this channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE) to the bath (10⁻⁷ M) abolished the response to NO. NO donors also increased the diameter of serotonin-preconstricted MCA to 85% of control. Blockade of K⁺ channels with iberiotoxin or a high-K⁺ medium reduced this response by 50%. ODQ (10⁻⁵ M) reduced this response by 47 ± 3%, whereas preventing the fall of 20-HETE levels reduced the response by 59 ± 2% (n = 5). Blockade of both pathways eliminated the response to NO donors. These results indicate that activation of K⁺ channels contributes 50% to vasodilator response to NO in rat MCA. This is mediated by a fall in 20-HETE levels rather than a rise in cGMP levels or a direct effect of NO.