The antinociceptive effect of huperzine A, a novel cholinesterase inhibitor, was investigated in the mouse hot-plate and abdominal constriction tests. Huperzine A induced a dose-dependent antinociception (70–110 mg kg–1 i.p.) which was prevented by scopolamine (0.1 mg kg–1 i.p.) and S-(–)-ET 126 (0.01 mg per mouse i.c.v.), but not by naloxone (1 mg kg–1 i.p.), mecamylamine (2 mg kg–1 i.p.), a- methyl-p-tyrosine (100 mg kg–1 i.p.), or CGP 35348 (100 mg kg–1 i.p.). A dose-dependent inhibition of the antinociception induced by huperzine A (110 mg kg–1 i.p.) was observed after inactivation of the M1 gene by an antisense oligodeoxyribonucleotide (aODN). This effect was detected 24 h after the last intracerebroventricular injection of aODN. Time-course experiments revealed that, after the end of the aODN treatment, sensitivity to analgesic drugs progressively appeared, reaching the normal range at 96 h. Huperzine A, at the maximal effective doses, did not produce any alteration of mice motor coordination, as revealed by rota-rod experiments. These results indicate that huperzine A is endowed by muscarinic antinociceptive properties mediated by the activation of central M1 muscarinic receptor subtype.