1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A. 5 In myocytes, the activation of adenylate cyclase caused by beta2-adrenoceptors was not detected in the presence of 10 nM, 100 nM or 1000 nM CGP 20712A, which selectively antagonized beta1-adrenoceptors.Furthermore, the concentration-response curve for isoprenaline-stimulated cyclic AMP accumulation was not shifted by 10 nm or 100 nM ICI 118,551, which selectively antagonized beta2-adrenoceptors, but was shifted to the right by 1000 nM ICI 118,551.6 These results indicate that beta2-adrenoceptors are not present on rat ventricular myocytes and that beta2-adrenoceptor stimulation does not cause any detectable production of cyclic AMP. We conclude that only beta1-adrenoceptors exist on rat ventricular myocytes.