The muscarinic-gated atrial potassium (IKAch) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. IKACh is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of IKACh, the contribution of Kir3.1 to IKaCh is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cell-attached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate IKACh. We conclude that Kir3.1 confers properties to IKACh that enhance channel activity and that Kir3.4 bomomultimers do not contribute significantly to the muscarinic-gated potassium current.