We have used the pilocarpine-induced seizure model in mice and i.c.v. injection of subtype-specific receptor antagonists to investigate the muscarinic receptor subtype specificity of cholinergically-activated seizures. The rank order potencies of antagonists for inhibition of pilocarpine-induced seizures are atropine = telenzepine > 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP) > pirenzepine with ID50's of 8.6, 12.0, 29.9, and 83.0 nmol/mouse, respectively. The M3-specific antagonists hexahydrosila-difenidol and its p-fluoro analog showed no effect on pilocarpine-induced seizures. The M2-specific antagonists gallamine and methoctramine cause seizures in mice in the absence of a pilocarpine injection. These seizures could be inhibited by coinjection of methoctramine with the M1-specific antagonist, pirenzepine. These data suggest a role of muscarinic M1 receptors in mediating pilocarpine-induced seizures and a role of the muscarinic M2 receptors in modulating neuronal activity.