Cultured human corneal epithelial cells showed high-affinity, specific binding to the muscarinic cholinergic antagonist, 3H-quinuclidinyl benzilate (3H-QNB). The binding sites had a dissociation constant of 3.9 nM and a maximal binding capacity of 880 fmol bound/mg protein. Other muscarinic antagonists (cyclopentolate and atropine) effectively competed for binding with 3H-QNB at low concentrations (IC50 = 10 nM). The muscarinic cholinergic agonist carbachol also competed for binding with 3H-QNB at somewhat higher concentrations (IC50 = 0.5 microM), and the nicotinic cholinergic agonist, nicotine, was at least 400-fold less potent than carbachol. Carbachol stimulated cyclic guanosine monophosphate (GMP) levels in these cells up to threefold over control. This stimulation was sensitive to atropine inhibition, requiring only 2 nM atropine for 50% inhibition and 100 nM of atropine to block the carbachol effect completely. A 90% decrease in specific 3H-QNB binding was observed if cultured cells were homogenized and fractionated before assay. Significant levels of specific 3H-QNB binding could also be observed when intact human corneas were incubated with 3H-QNB and their epithelium subsequently isolated before measurement of bound radioligand. These studies indicate the presence of muscarinic cholinoceptors in human corneal epithelium which are associated with control of cyclic GMP levels in this tissue.