Characterization of adrenergic receptors in membranes from the rat seminal vesicle was studied by radioligand binding assay. Seminal vesicle membranes contained saturable and high affinity binding sites for the beta-adrenergic receptor antagonist 3H-dihydroalprenolol (3H-DHA) and for the alpha-adrenergic antagonist 3H-prazosin. The observed order of potency for adrenergic agonists in competing for the 3H-DHA binding sites: isoproterenol > epinephrine congruent to salbutamol > norepinephrine indicates that these membrane receptors have the properties of beta 2-adrenergic receptors. alpha 1-Adrenergic receptors were defined mainly as alpha 1A subtypes by demonstrating their insensitivity to pretreatment with chlorethylclonidine and the different rank orders of antagonist affinities. No significant binding sites for the alpha 2-adrenergic receptor agonist 3H-clonidine were observed. The GTP-induced reduction in the affinity of alpha 1-adrenergic receptors for epinephrine was significantly reversed by the muscarinic cholinergic agonist carbachol. Atropine effectively antagonized this effect of carbachol on the competitive inhibition of 3H-prazosin binding by epinephrine in the presence of GTP, which suggests that muscarinic cholinergic receptors regulate the affinity of alpha 1-adrenergic receptors by modulating the effect of guanine nucleotides. The effect of GTP on decreasing the affinity of beta 2-adrenergic receptors was not influenced by the addition of carbachol.