Crude membrane preparations from the dorsomedial medulla and whole heart of adult rats were used to characterize muscarinic receptors (mChRs) in the nucleus tractus solitarius (NTS). Isotherm binding assays with [3H]quinuclidinyl benzilate ([3H]QNB) indicated a uniform population of muscarinic cholinergic receptors in both tissues with a Kd of 60.3 pM and a Bmax of 401 fmol/mg protein for NTS membranes while heart membranes had a Kd of 56.6 pM and a Bmax of 193 fmol/mg protein. Competitive binding assays with selective muscarinic antagonists on NTS membranes using 80 pM [3H]QNB showed the following relative Ki values consistent with M2 mChRs: atropine, 1.9 nM; methoctramine, 7.7 nM; 4-diphenylacetoxy-N-methylpiperidine methobromide, 9.4 nM; AF-DX 116, 87.6 nM; and pirenzepine, 657 nM. Similarly, two agonists, carbachol and oxotremorine, showed Ki values of 1.82 microM and 0.379 microM, respectively. All of the competitive binding assays exhibited Hill coefficients close to 1.0 and InPlot analysis showed a single binding site for each agonist and antagonist examined.