Specific and high affinity binding of the potent muscarinic cholinergic antagonist, [3H] quinuclidinylbenzilate ([3H] QNB) was observed using intact native and cultured adult human corneal endothelium (HCE). Specific binding was proportional to radioligand concentration between 0.03 and 5 nM, indicating a maximal binding capacity (Bmax) of 130 fmol of [3H] QNB/mg protein and a dissociation constant (Kd) of 0.3 nM. Atropine competed effectively with [3H] QNB for binding sites; requiring 3 nM to inhibit 50% of the binding of 1 nM [3H] QNB. Carbachol also competed with [3H] QNB at higher concentrations, but nicotine did not affect [3H] QNB binding at levels up to 1 mM. [3H] QNB binding was also observed in cultured cells of adult human, rabbit, and bovine corneal endothelium.Native and cultured HCE were affinity labelled using tritium-labelled propylbenzilylcholine mustard (PBCM). Separation of the proteins in affinity labelled native and cultured tissue by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) showed that only one protein in each preparation, of 60 and 55 kilodaltons (kDa), respectively, was specifically radiolabelled.These data indicate that the corneal endothelium of human and several animal species exhibit muscarinic cholinoceptors.