Members of the Kir2 subfamily of inwardly rectifying K+ channels characterized by their strong current rectification are widely expressed both in the periphery and in the CNS in mammals. We have cloned from rat brain a fourth subfamily member, designated Kir2.4 (IRK4), which shares 53-63% similarity to Kir2.1, Kir2.2, or Kir2.3 on the amino acid level. In situ hybridization analysis identifies Kir2.4 as the most restricted of all Kir subunits in the brain. Kir2. 4 transcripts are expressed predominantly in motoneurons of cranial nerve motor nuclei within the general somatic and special visceral motor cell column and thus are uniquely related to a functional system. Heterologous expression of Kir2.4 in Xenopus oocytes and mammalian cells gives rise to low-conductance channels (15 pS), with an affinity to the channel blockers Ba2+ (Ki = 390 microM) and Cs+ (Ki = 8.06 mM) 30-50-fold lower than in other Kir channels. Low Ba2+ sensitivity allows dissection of Kir2.4 currents from other Kir conductances in hypoglossal motoneurons (HMs) in rat brainstem slices. The finding that Ba2+-mediated block of Kir2.4 in HMs evokes tonic activity and increases the frequency of induced spike discharge indicates that Kir2.4 channels are of major importance in controlling excitability of motoneurons in situ.