The muscarinic receptor subtype present in the bovine adrenal medulla was characterized. Hybridization of RNA to highly specific m1-m5 muscarinic receptor cDNA probes detected the presence of only m4 subtype mRNA in this tissue. Muscarinic receptor binding studies using the non-selective ligand [3H]N-methyl-scopolamine showed a single class of binding sites with a maximum density of 19.8 fmol/mg protein and a dissociation constant (KD) of 220 pM in the adrenal medulla, while the M1 selective ligand [3H]telenzepine did not bind detectably. Competition of specific antagonists with [3H]N-methyl-scopolamine for binding to the membranes produced a rank order of potencies with a profile that fitted either the cloned m3 or m4 receptor. In further comparative studies, the adrenal gland of the rat showed the presence of m4 subtype mRNA in addition to the m3 subtype.