Binding of [3H]-pilocarpine to synaptic membranes in rat cerebral cortex was investigated, pilocarpine binding was also studied by competition of unlabelled pilocarpine with the [3H]-labelled muscarinic antagonist, [3H]-N-methyl-4-piperidinyl benzilate. 1. [3H]-pilocarpine binding sites are of protein nature, and the highest specific activity of binding is found in the synaptosomal fraction of all subcellular fractions. 2. Competition studies show that only muscarinic drugs inhibit [3H]-pilocarpine binding in their pharmacologically active concentration range. 3. Binding of [3H]-pilocarpine is influenced by GMPP-(NH)P (0.1 mM) similarly to the binding of other muscarinic agonists. 4. Examination of pilocarpine binding with [3H]-pilocarpine and the competition experiments with unlabelled pilocarpine indicate the presence of three sites with different affinities: 5 nM, 0.2 microM and 30 microM respectively. 5. Experiments with [3H]-4-NMPB and [3H]-pilocarpine indicate that there are more [3H]-pilocarpine binding sites than [3H]-4-NMPB binding sites in rat cerebral cortex.