We have investigated the acute desensitization of acetylcholine-activated GIRK current (IK(ACh)) in cultured adult rat atrial myocytes. Acute desensitization of IK(ACh) is observed as a partial relaxation of current with a half time of < 5 s in the presence of the agonist when muscarinic M2 receptors are stimulated by a high concentration (>2 µmol/L) of ACh. Under this condition experimental manoeuvres that cause a reduction in the amplitude of IK(ACh) such as partial block of M2 receptors by atropine, intracellular loading with GTP-b-S, or exposure to Ba2+ caused a reduction in desensitisation. Acute desensitisation was also identified as a reduction in current amplitude and a blunting of the response to saturating [ACh] (20 µmol/L) when the current has been partially activated by a low concentration of ACh or by stimulation of A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP-dependent K+ current (IK(ATP)) was activated either by mitochondrial uncoupling using DNP or by the channel opener rilmakalime. Adenovirus-driven overexpression of Kir2.1, a subunit of constitutively active inwardly-rectifying K+ channels, resulted in a large Ba2+-sensitive background K+ current and a dramatic reduction of ACh-activated current. Adenovirus-driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist-independent GIRK current paralleled by a reduction in IK(ACh) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than a bona fide signalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential of IK(ACh). Since the ACh-induced hyperpolarization shows comparable desensitization properties as IK(ACh), this novel current-dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.