The muscarinic K(+) channel (I (K,ACh)) is a heterotetramer composed of GIRK1 (Kir3.1) and GIRK4 (Kir3.4) subunits of a G protein-coupled inwardly rectifying channel, and plays an important role in mediating electrical responses to the vagal stimulation in the heart. I (K,ACh) displays biphasic changes (activation followed by inhibition) through the stimulation of the purinergic P2Y receptors, but the regulatory mechanism involved in these modulation of I (K,ACh) by P2Y receptors remains to be fully elucidated. Various P2Y receptor subtypes and GIRK1/GIRK4 (I (GIRK)) were co-expressed in Chinese hamster ovary cells, and the effect of stimulation of P2Y receptor subtypes on I (GIRK) were examined using the whole-cell patch-clamp method. Extracellular application of 10 μM ATP induced a transient activation of I (GIRK) through the P2Y(1) receptor, which was completely abolished by pretreatment with pertussis toxin. ATP initially caused an additive transient increase in ACh-activated I (GIRK) (via M(2) receptor), which was followed by subsequent inhibition. This inhibition of I (GIRK) by ATP was attenuated by co-expression of regulator of G-protein signaling 2, or phosphatidylinositol-4-phosphate-5-kinase, or intracellular phosphatidylinositol 4,5-bisphosphate loading, but not by the exposure to protein kinase C inhibitors. P2Y(4) stimulation also persistently suppressed the ACh-activated I (GIRK). In addition, I (GIRK) evoked by the stimulation of the P2Y(4) receptor exhibited a transient activation, but that evoked by the stimulation of P2Y(2) or P2Y(12) receptor showed a rather persistent activation. These results reveal (1) that P2Y(1) and P2Y(4) are primarily coupled to the G(q)-phospholipase C-pathway, while being weakly linked to G(i/o), and (2) that P2Y(2) and P2Y(12) involve G(i/o) activation.