The cardiac inward rectifying K⁺ current (I _K1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricularI _K1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents inXenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4–5 days culture), these oligonucleotides caused a significant reduction of whole cell I _K1(without reducing the transient outward K⁺ current or the L-type Ca²⁺ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K⁺-channel protein and that this channel has an essential role in the genesis of cardiacI _K1.