This study evaluates β-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [3H]-CGP12177 selectively bound to plasma membrane β-adrenoceptors, and the Bmax levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of β1- and β2-adrenoceptors. The regional distribution of plasma membrane β1- and β2-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of β-adrenoceptors and proportion of β1-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study β-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (−)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (−)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of β-adrenoceptors, predominantly β2-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (−)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [3H]-CGP12177 enables a more precise quantitation of plasma membrane β1- and β2-adrenoceptors in rat hearts and is suitable for studying their regulation.