Selective ligands are needed for distinguishing the functional roles of M2 receptors in tissues containing several muscarinic receptor subtypes. Because the venom of the green mamba Dendroaspis angusticeps contains the most specific antagonists known for M1 and M4 receptors (m1-toxin and m4-toxin), it was screened for toxins that inhibit the binding of [(3)H]N-methylscopolamine ([(3)H]NMS) to cloned M2 receptors. Desalted venom had as much anti-M2 as anti-M4 activity. The most active anti-M2 toxin in the venom was isolated by gel filtration, cation-exchange chromatography, and reversed-phase HPLC, and called m2-toxin. Spectrometry yielded a mass of 7095 Da, and N-terminal sequencing of 53 amino acids showed RICHSQMSSQPPTTTFCRVNSCYRRTLRDPHDPRGT-IIVRGCGCPRMKPGTKL. This sequence is more homologous to antinicotinic than antimuscarinic toxins, but it lacks three almost invariant residues of antinicotinic toxins required for their activity. m2-Toxin fully blocked the binding of [(3)H]NMS and [(3)H]oxotremorine-M to M2 receptors with Hill coefficients near 1, and blocked 77% of the binding sites for 0.1 nM [(3)H]NMS in the rat brainstem (K(i) = 11 nM). Concentrations that fully blocked cloned M2 receptors had no effect on M4 receptors, but slightly increased [(3)H]NMS binding to M1 receptors, an allosteric effect. In accord with these results, light microscopic autoradiography of the rat brain showed that m2-toxin decreased [(3)H]NMS binding in regions rich in M2 receptors and increased binding in regions rich in M1 receptors. Thus m2-toxin is a novel M2-selective, short-chain neurotoxin that may prove useful for binding and functional studies.