The ATP-sensitive potassium (K(ATP)) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal K(ATP) channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal K(ATP) channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type K(ATP)) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial K(ATP) channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H(2)O(2))-decomposing enzyme. These reagents also ablated NO-induced K(ATP) channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H(2)O(2) reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H(2)O(2) was able to enhance the function of K(ATP) channels in the presence of Ca(2+) chelators and calmodulin antagonists, whereas the stimulatory effect of H(2)O(2) was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal K(ATP) channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca(2+)/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal K(ATP) channels.