<jats:p> <jats:italic>Background</jats:italic> The present study investigated biochemical and functional consequences of chronic activation of the inhibitory G <jats:sub>iα</jats:sub> -coupled adenylyl cyclase pathway in the heart. </jats:p> <jats:p> <jats:italic>Methods and Results</jats:italic> Rats (220 to 260 g) were treated with 4-day infusions of the M-cholinoceptor agonist carbachol (9.6 mg/kg per day) or vehicle. An additional group that received the β-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) served as control. The main finding was that chronic infusion of carbachol led to a marked increase in isoprenaline- or forskolin-induced arrhythmia in electrically driven papillary muscles (in vitro). Compared with control, the potency of isoprenaline and forskolin to induce arrhythmia in cardiac preparations from carbachol-treated rats was increased 36- and 2.2-fold and the efficacy was increased 7.3- and 2.3-fold, respectively. The potency of carbachol to antagonize the isoprenaline- and forskolin-induced arrhythmia was decreased 30-fold. These changes were accompanied by a decrease in left ventricular M-cholinoceptor density by 15% ( <jats:italic>P</jats:italic> <.05) and a decrease in pertussis toxin–sensitive G proteins (G <jats:sub>iα</jats:sub> ) by 26% ( <jats:italic>P</jats:italic> <.05) without a decrease in the corresponding mRNAs. β-Adrenoceptor density and basal and stimulated adenylyl cyclase activity remained unchanged. In contrast, isoprenaline infusion induced a decrease in arrhythmogenic potency of forskolin ( <jats:italic>P</jats:italic> =NS), which was accompanied by a decrease in β-adrenoceptor density, an increase in G <jats:sub>iα</jats:sub> protein and mRNA levels, and a decrease in basal and stimulated adenylyl cyclase activity. </jats:p> <jats:p> <jats:italic>Conclusions</jats:italic> Chronic parasympathetic activation sensitizes the myocardium to cAMP-induced arrhythmia. These changes may be due to quantitative alterations in functional G <jats:sub>iα</jats:sub> . </jats:p>