Abstract The expression of three different subtypes of GTP binding protein β-subunits (β 1, β 2 and β 3) was examined in rat hearts at both mRNA and protein levels. Among three subtypes, β 3 mRNA was more abundant in ventricles while β 1 and β 2 mRNAs were expressed ubiquitously among ventricles, atria and aortas. The immunodetectable β 1- and β 3-proteins were predominantly found in the cytosolic fraction of rat ventricles. In this study, we demonstrate ventricle-specific expression of β 3-subunit and localization of G protein β 3-subunit in the cytosol of rat hearts. This unusual cytosolic localization of G protein β 3-subunit might indicate subtype-specific function of G protein β(γ)-subunits in rat hearts.