In atrial and nodal cardiac myocytes binding of acetylcholine to M2 muscarinic receptors activates inhibitory G-proteins which in turn stimulate G-protein-gated inward rectifying K+ channels (GIRK1/GIRK4) through direct binding of the Gβγ-subunit. In contrast, other Gβγ releasing receptors e.g. Gs coupled β-adrenergic receptors are not able to activate this current. An appealing hypothesis to explain these observations would be if components were sequestered in membrane domains such as rafts/caveolae. Methods: Standard biochemical fractionation techniques followed by Western blotting and/or radioligand binding experiments were used to examine the signalling components in HEK293 and HL-1 (mouse atrial cardiomyocyte cell line) cells which natively express this transduction cascade. In addition, expression and localisation were analysed by confocal and Total Internal Reflection Fluorescence (TIRF) microscopy. Whole-cell patch clamp was used to study the activation kinetics of the GIRK current. Results: Western Blot analysis (n=3) showed that the channel (GIRK1/GIRK4) and M2 receptors were located in noncaveolar fractions. However, Gαi was enriched in both, caveolar fractions and noncaveolar fractions. In contrast, Gαs was only enriched in caveolar fractions. Radioligand binding experiments (n≥3) confirmed M2 and A1 receptor localisation in noncaveolar fractions. To further explore the potential function of caveolae a YFP tagged variant of caveolin-2 (YFP-Cav2) and receptor chimaeras fused to YFP-Cav2, M2- (M2-YFP-Cav2) and A- (A1-YFP-Cav2) receptors were constructed. The analysis of gradient fractions of receptor-YFP-Cav2 expressing cells showed that the receptors were now localised in caveolar fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse and homogenous membrane signal. YFP-Cav2 revealed a much more punctuate pattern corresponding to membrane domains or vesicles present just below the membrane. M2-YFP-Cav2 and A1-YFP-Cav2 showed a similar pattern as YFP-Cav2 albeit with less efficient targeting. Functional studies revealed slower current activation when activated through the caveolae targeted A1 or M2 receptors, reflected by an increase in the lag + TTP and τac for the M2 receptors (P<0.05, Kruskal-Wallis test, used with a Dunn post-hoc test). Conclusion: In HEK293 and HL-1 cells the GIRK transduction cascade is localised outside of caveolae. It is therefore possible that by localising Gαi within caveolae the activity of the signalling pathway could be altered. The localisation of Gαs solely in caveolae may also provide a mechanism for selectivity. The localisation in caveolar domains of the GPCR involved in the GIRK signal transduction cascade substantially slows the speed of signalling.