Acute opioid administration causes hyperpolarization of locus ceruleus (LC) neurons. A G-protein-gated, inwardly rectifying potassium (GIRK/K(G)) conductance and a cAMP-dependent cation conductance have both been implicated in this effect; the relative contribution of each conductance remains controversial. Here, the contribution of K(G) channels to the inhibitory effects of opioids on LC neurons was examined using mice that lack the K(G) channel subunits Kir3.2 and Kir3.3. Resting membrane potentials of LC neurons in brain slices from Kir3.2 knock-out, Kir3.3 knock-out, and Kir3.2/3.3 double knock-out mice were depolarized by 15-20 mV relative to LC neurons from wild-type mice. Metenkephalin-induced hyperpolarization and whole-cell current were reduced by 40% in LC neurons from Kir3.2 knock-out mice and by 80% in neurons from Kir3.2/3.3 double knock-out mice. The small opioid-sensitive current observed in LC neurons from Kir3.2/3.3 double knock-out mice was virtually eliminated with the nonselective potassium channel blockers barium and cesium. We conclude that the acute opioid inhibition of LC neurons is mediated primarily by the activation of G-protein-gated potassium channels and that the cAMP-dependent cation conductance does not contribute significantly to this effect.