Chemical modification of muscarinic M1 receptors in a synaptoneurosomal preparation of rat cerebral cortex by a hydrophilic histidyl-group-specific reagent, diethylpyrocarbonate (DEP), reduces the number of [3H]-4NMPB binding sites in a dose-dependent way. The effect can be reversed by hydroxylamine treatment. No such effect is observed when carbethoxylation with 2.5 mM DEP is carried out in the presence of atropine, 4NMPB, pirenzepine or carbachol. These findings indicate that DEP specifically modifies histidyl residue(s) positioned at the binding site in members of the M1 receptor family. However, treatment with 2.5 mM DEP in the presence of various muscarinic ligands significantly disturbs the binding state of agonists. The results suggest that M1 receptors may have more than one histidyl residue of importance in ligand binding.