The binding characteristics of [3H]quinuclidinyl benzilate ([3H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [3H]QNB bound to endothelial cell membranes with high affinity (kD = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (Ki) were determined by displacement of bound [3H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M3 antagonist) > pirenzepine (M1 antagonist) > AFDX-116 (M2 antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M1 agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M3 and M1 subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [3H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [3H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.