The hydrophobic core of all muscarinic receptors contains several conserved serine, threonine and tyrosine residues, most of which do not occur in any other G-protein coupled receptor. Since these amino acids can serve as potential hydrogen bond donors or acceptors, we have tested the hypothesis that they may be involved in the selective binding of muscarinic ligands. To eliminate the OH groups present in these residues, we have created nine single point mutations in the rat m3 muscarinic receptor by converting serine and threonine residues to alanine, and tyrosine residues to phenylalanine. The ligand binding and functional properties of these receptors were studied after transient expression in COS-7 cells. Six out of the nine mutant receptors (threonine and tyrosine mutations) showed strong reductions (approximately 10- to 40-fold lower than the wild-type receptor) in agonist binding affinities and reduced potencies in agonist-induced activation of phosphoinositide hydrolysis. Their antagonist binding properties, however, were similar to those of the wild-type m3 receptor. Despite their location on different transmembrane domains (III, V, VI and VII), all six mutations are positioned at a similar level (one to two helical turns away from the membrane surface) within the outer leaflet of the plasma membrane and may thus define the plain in which muscarinic agonists (but not antagonists) bind to their target receptor.