Learning and memory are critically dependent on the excitability of basal forebrain cholinergic (BFC) neurons, which is modulated profoundly by leak K⁺ channels. Many neuromodulators closing leak K⁺ channels have been reported, whereas their endogenous opener remained unknown. We here demonstrate that nitric oxide (NO) can be the endogenous opener of leak K⁺ channels in the presumed BFC neurons. Bath application of 1 mM S-nitroso-N-acetylpenicillamine (SNAP), an NO-donor, induced a long-lasting hyperpolarization, which was often interrupted by a transient depolarization. Soluble guanylyl cyclase inhibitors prevented SNAP from inducing hyperpolarization but allowed SNAP to cause depolarization, while bath application of 0.2 mM 8-Br-cGMP induced a similar long-lasting hyperpolarization alone. These observations indicate that the SNAP-induced hyperpolarization and depolarization are mediated by the cGMP-dependent and -independent processes, respectively. When examined with the ramp command pulse applied at –70 mV under the voltage-clamp condition, 8-Br-cGMP application induced the outward current that reversed at K⁺ equilibrium potential (E_K) and displayed Goldman-Hodgkin-Katz rectification, indicating the involvement of voltage-independent K⁺ current. By contrast, SNAP application in the presumed BFC neurons either dialyzed with the GTP-free internal solution or in the presence of 10 µM Rp-8-Br-PET-cGMPS, a PKG inhibitor, induced the inward current that reversed at potentials much more negative than E_K and close to the reversal potential of Na⁺-K⁺ pump current. These observations strongly suggest that NO activates leak K⁺ channels through cGMP-PKG-dependent pathway to markedly decrease the excitability in BFC neurons, while NO simultaneously causes depolarization by the inhibition of Na⁺-K⁺ pump through ATP depletion.