1. 1. Binding sites in guinea pig myocardial tissue labelled by (-)-[125I]cyanopindolol (CYP) were investigated using differential centrifugation and autoradiographic techniques. Autoradiographs of myocardial sections (0.1 μm) indicated (-)-[125I]CYP binding to sarcolemmal membrane. A low density of binding sites was observed to mitochondria. 2. 2. Binding studies were performed in subcellular fractions. The density of binding sites in the mitochondrial fraction (36.1 ± 9.4 fmol/mg protein) was <10% that in the sarcolemmal membrane (371.7 ± 38.2 fmol/mg protein). The β1/β2-adrenoceptor subtype ratio in the mitochondrial fraction (83.3/16.7) was similar to that in the sarcolemmal fraction (87.1/12.9). 3. 3. Ouabain (100 μM), in the presence of sodium azide (0.4 mM), inhibited a Na+K+ stimulated ATPase activity (1.0 ± 0.2 μmol Pi/mg protein/hr reduction), indicating a low but significant level of sarcolemmal contamination of the mitochondrial fraction. 4. 4. The study showed β-adrenoceptors in guinea pig heart are located primarily on the sarcolemmal membrane of myocardium. No evidence was obtained for β-adrenoceptors over mitochondria, as has been suggested in other tissues and species, but that this binding was to sarcolemmal inclusions in the mitochondrial fraction.