Objective: In atrial and nodal cardiac myocytes binding of acetylcholine to M2 muscarinic receptors activates inhibitory G-proteins which stimulate G-protein-gated inward rectifying K+ channels (GIRK1\GIRK4) through direct binding of the Gbg-subunit. Also releasing Gbg e.g. Gs coupled b-adrenergic receptors are not able to activate this current. An appealing hypothesis to explain these observations would be if components are sequestered in membrane domains such as caveolae\rafts. Methods: Standard biochemical fractionation techniques followed by Western blotting and\or radioligand binding experiments where used to examine the signalling components in HEK293 and HL-1 cells (mouse atrial cardiomyocyte cell line) which natively express the transduction cascade. Expression and localisation were analysed by confocal microscopy as well as total internal reflection fluorescence (TIRF) microscopy. Results: The channel (GIRK1\GIRK4) and both M2 and A1 receptors were located in noncaveolar fractions in both cell lines. However, Gai was enriched in caveolar fractions but was also present in noncaveolar domains. In contrast Gas was only enriched in caveolar fractions. The addition of an agonist did not alter this distribution. Gradient fractions of cells expressing YFP tagged receptors fused to Caveolin-2 (Cav-2) showed that the receptors are 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 but with less efficient targeting. Conclusion: In HEK293 and HL-1 cells the GIRK transduction cascade is localised outside of caveolae. It is therefore possible that by localising Gi within caveolae the activity of the signalling pathway could be altered. The localisation of Gas solely in caveolae may be a selectivity mechanism.