Summary Recent studies suggest that two subtypes of a-adrenergic receptors (α1 and α2) can be distinguished on the basis of the differential affinities of certain adrenergic ligands for each subtype. We have investigated the binding characteristics of several such ligands for α-adrenergic receptors in membrane preparations derived from rat heart. α-Adrenergic antagonists competed for binding sites identified by the nonsubtype selective α-adrenergic ligand [3H]dihydroergocryptine (DHE) in the order of potency expected for α1-receptors, namely, prazosin (EC50 = 0.35 nM) > phentolamine (EC50) = 37 nM) > yohimbine (EC50 = 918 nM). Furthermore, the nonsubtype selective radioligand [3H]DHE identified a quantitatively similar number of specific binding sites in rat cardiac membranes as the α2-selective radioligand [3H]prazosin (33 and 36 fmoles/mg protein, respectively), while the α2-selective ligand [3H]clonidine at concentrations up to 20 nM demonstrated negligible specific binding. We conclude that the α-adrenergic receptors of rat heart homogenates demonstrate binding characteristics typical of α2-receptors. While we cannot exclude the presence of small numbers of α2-receptors, the similar number of binding sites identified at saturation for the α1-specific ligand [3H]prazosin and for the nonsubtype selective ligand [3H]DHE supports the hypothesis that the a-receptors of rat heart are predominantly of the α1-subtype.