The striking seasonal difference in sensitivity of frog cardiac muscle to acetylcholine or stimulation of parasympathetic nervous fibers has been noted almost a century ago, although its electrophysiological basis has never been revealed. The present study compares the effects of the muscarinic agonist carbamylcholine chloride (CCh 10-8-10-5M) on electrical activity in isolated atrial and ventricular preparations from dormant frogs (Rana temporaria) caught in January (winter-acclimatized, WA) and from active frogs caught in July (summer-acclimatized, SA). Seasonal differences in the density of potassium acetylcholine-dependent current (IKACh) were also studied in atrial and ventricular myocytes from both summer and winter groups. In atrial myocardium, CCh produced concentration-dependent shortening of action potentials (APs). CCh concentration producing a 50% reduction of AP duration was lower in WA (1.03 × 10-7M) than SA atria (2.7 × 10-7M). 10-6M CCh induced drastic reduction of AP amplitude rendering the tissue unexcitable in both WA or SA atrial preparations. Ventricular preparations showed greater seasonal difference in CCh sensitivity. While 10-6M induced inexcitability in 50% of tested WA preparations, in SA preparations even 10-5M CCh was without effect. This striking difference between cholinergic effects in SA and WA frog ventricle could be explained by seasonal changes in the IKACh density. The density of IKACh induced by 10-5M CCh and measured at 0mV was 14.4 ± 3.45pA/pF in WA, but only 1.5 ± 0.4pA/pF in SA atrial cells. In ventricular cells, the respective values were 2.61 ± 0.56 and 0.71 ± 0.09pA/pF. Thus, hibernating winter frog has a much greater electrophysiological cholinergic response than active summer frog due to up-regulation of IKACh.