We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M₁-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M₁-M₅ receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M₁-M₅ receptor single KO mice. In M₂ and M₄ receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M₂ receptor-immunoreactivity was located in both smooth muscle cells and enteric neurons. M₄ receptor-immunoreactivity was located in the enteric neurons, being in colocalization with M₂ receptor-immunoreactivity. These results indicate that both M₂ and M₄ receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. Neither M₁, M₃, nor M₅ receptors appear to be involved in this mechanism.