ATP-sensitive K⁺ channels play an important role in coupling membrane excitability with intracellular metabolic stress. To characterize such K_ATP channels from rat brain, the inside-out mode of patch-clamp technique was applied to freshly dissociated hippocampal CA1 pyramidal neurons of adult rat. One type of K⁺ permeable channel was recorded only at the presence of Ca²⁺ in the internal solution and it could be inhibited by application of 1～3 mmol/L ATP and 1 mmol/L tolbutimade, a K_ATP channel blocker. Both of channel open probability and the ATP induced-inhibition displayed a voltage-dependent fashion. When both sides of the excised membrane were in symmetrical 140 mmol/L K⁺, the I-V relation was linear with a conductance of 204 pS and reversal potential was 3.57 mV. Unlike the previously reported “classical” K_ATP channel, this large-conductance K_ATP channel (L-K_ATP) was regulated by membrane potential, intracellular Ca²⁺ and ATP, indicating a new subtype of K_ATP channel presents in hippocampus neurons. These results demonstrate that at least two distinct K_ATP channels exist in rat hippocampal neurons and suggest that metabolic state may be continuously sensed in neurons via different K_ATP channels with resulting alterations in neuronal membrane excitability.