As a first step in understanding the functioning of hormonal signaling pathways in the heart, the G protein composition of neonatal and adult rat hearts was determined by immunoblotting analysis. Neonatal rat cardiac myocytes and nonmuscle heart cells contained Gs alpha 52, Gi alpha 1,3, and Gi alpha 2 with little or no Gs alpha 45 or Go alpha 39. Interestingly, Go alpha 39 accumulated fourfold in cardiac myocytes between days 1 and 6 of culture. Atrial membranes from adult rat hearts had approximately equal amounts of the 45- and 52-kDa forms of Gs alpha, whereas adult ventricles had predominantly Gs alpha 45. In addition, adult atria contained relatively more Gi alpha 1,3, Gi alpha 2, and Go alpha 39 than adult ventricle. Moreover, the increase in Go alpha 39 observed in cardiac myocytes cultured for 6 days could be prevented by culturing cells in medium containing 50 mM KCl. The observed differences in G protein expression between cell types and between contracting and KCl-depolarized cardiac myocytes may provide a system to investigate the function of various G proteins and to study regulation of their expression.