AbstractMuscarinic agonist‐induced parasympathomimetic effects, in vivo phosphoinositide hydrolysis and seizures were evaluated in wild‐type and muscarinic M1–M5 receptor knockout mice. The muscarinic agonist oxotremorine induced marked hypothermia in all the knockout mice, but the hypothermia was reduced in M2 and to a lesser extent in M3 knockout mice. Oxotremorine‐induced tremor was abolished only in the M2 knockout mice. Muscarinic agonist‐induced salivation was reduced to the greatest extent in M3 knockout mice, to a lesser degree in M1 and M4 knockout mice, and was not altered in M2 and M5 knockout mice. Pupil diameter under basal conditions was increased only in the M3 knockout mice. Pilocarpine‐induced increases in in vivo phosphoinositide hydrolysis were completely absent in hippocampus and cortex of M1 knockout mice, but in vivo phosphoinositide hydrolysis was unaltered in the M2–M5 knockout mice. A high dose of pilocarpine (300 mg/kg) caused seizures and lethality in wild‐type and M2–M5 knockout mice, but produced neither effect in the M1 knockout mice. These data demonstrate a major role for M2 and M3 muscarinic receptor subtypes in mediating parasympathomimetic effects. Muscarinic M1 receptors activate phosphoinositide hydrolysis in cortex and hippocampus of mice, consistent with the role of M1 receptors in cognition. Muscarinic M1 receptors appear to be the only muscarinic receptor subtype mediating seizures.