Preliminary evidence of muscarinic acetylcholine receptors (AchRs) in rabbit and human peritoneal mesothelial cells grown in tissue culture is reported. Atropine displaceable binding of L-quinuclidinyl[phenyl-4-3H]-benzilate (QNB), an antagonist that binds to all AchR subtypes, and [N-methyl-3H]-4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), an antagonist specific for AchR subtypes 1 and 3, to homogenates of mesothelial cells was approximately 400 and 120 fmol ligand bound/milligram cell protein, respectively. A similar value for specific [3H]-QNB binding was observed in living cells exposed to ligand and atropine for 1 hour at 37C. Guanosine 5'-triphosphate (GTP)-binding proteins measured as beta,gamma-imido[8-3H] guanosine 5'-triphosphate binding to mesothelial cell membranes was approximately 9 pmol/mg cell protein. Our finding of both muscarinic receptors and GTP-binding proteins in mesothelial cells suggests first, the presence of a functional muscarinic receptor system(s) in peritoneal mesothelial cells, and second, the potential susceptibility of these cells to direct pharmacological manipulation by muscarinic drugs.