The biochemical and physiological effects of G_Sα activation are well known; however, little is known about the anatomical localisation of G_Sα in the myocardium. Knowledge of the localisation might yield insights into G protein function in heart. The utility of immunocytochemistry using immunofluorescent methods is limited in normal hearts because of the low expression of G_Sα. In order to magnify the G_Sα signal, we studied transgenic mice overexpressing myocardial G_Sα. Immunofluorescent techniques with confocal imaging using rabbit antiserum specific for G_Sα were studied in frozen sections of mouse left ventricle. G_Sα labeling appeared to be localised to the T-tubules and intercalated disks in the G_Sα overexpressing mouse hearts, whereas the control mice showed background fluorescence with diffuse faint labeling. The localisation of G_Sα to structures involved in calcium handling and membrane conductance places G_Sα at a focal point in the regulation of these key functions.