Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K⁺ channel subfamily encode G protein-gated inwardly rectifying K⁺ channels (I_KACh) in other tissues, including the heart and brain. In the GI tract, I_KACh could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to -actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 µM) reversibly activated a Ba²⁺-sensitive and inwardly rectifying K⁺ current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I_KACh current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.