Binding studies with (−)-[125I]cyanopindolol (ICYP) were conducted to characterize β-adrenoceptors in plantaris and soleus muscles of rats (male, 250–300 g). The distribution of β1- and β2-adrenoceptors in different muscle fiber types, identified in serial sections by succinic dehydrogenase (SDH) staining, was studied by autoradiography. The densities of binding sites (Bmax, mol/mg protein) were 5.4 ± 0.9 (mean ± SEM) in plantaris and 11.5 ± 2.0 in soleus muscle. In plantaris muscle, monophasic competition curves were observed when binding experiments were performed using CGP 20712A (50 pM to O.SmM), a β1-adrenoceptor selective antagonist, or ICI 118,551 (50 pM to 20,μM), a β2-adrenoceptor selective antagonist, to compete for ICYP binding. Analysis with LIGAND revealed a single binding site with a KD value of 2.41 ± 0.56 nM (mean ± SEM) for ICI 118,551 and 8.93 ± 3.00 μM for CGP 20712A, indicating the presence of a homogeneous population of β2-adrenoceptors. In soleus muscle, competition curves were biphasic with 16–21% β1-adrenoceptors. Autoradiographic studies supported the findings from binding studies with membrane homogenates. The ICYP binding pattern was associated closely with the muscle fiber types identified by SDH staining. Propranolol-resistant binding sites were observed, and these sites were associated with muscle fibers positive to SDH staining.