Heterotrimeric G proteins are comprised of a guanine nucleotide binding Galpha subunit and the Gbetagamma dimers that link G protein-coupled receptors (GPCRs) to effectors. This study focuses on the expression and localization patterns for certain Gbeta and Ggamma subunits in neonatal and adult cardiomyocytes. We identify developmental downregulation of Gbeta1, Gbeta2, and Ggamma2, and a switch in the molecular form of Ggamma3, in cardiomyocytes. Gbeta1 is highly localized to caveolae membranes, whereas Gbeta2 is identified in caveolae and other membrane fractions. Gbeta3 is not detected in neonatal cardiomyocytes, but rather Gbeta3 is upregulated in adult cardiomyocytes and detected in the caveolae and soluble fractions. The observation that cardiomyocytes co-express multiple Gbeta and Ggamma subunits in a developmentally regulated manner, and that these Gbeta and Ggamma subunits assume distinct subcellular localization patterns, provides for a high level of signaling specificity in the heart.