Activation of spinal muscarinic acetylcholine receptors (mAChRs) produces analgesia and inhibits dorsal horn neurons through potentiation of GABAergic/glycinergic tone and inhibition of glutamatergic input. To investigate mAChR subtypes involved in the inhibitory effect of mAChR agonists on glutamate release, evoked excitatory postsynaptic currents (EPSCs) were recorded in lamina II neurons using whole-cell recordings in rat spinal cord slices. The mAChR agonist oxotremorine-M concentration-dependently inhibited the monosynaptic and polysynaptic EPSCs elicited by dorsal root stimulation. Interestingly, oxotromorine-M caused a greater inhibition of polysynaptic EPSCs (64.7%) than of monosynaptic EPSCs (27.9%). In rats treated with intrathecal pertussis toxin, oxotremorine-M failed to decrease monosynaptic EPSCs but still partially inhibited the polysynaptic EPSCs in some neurons. This remaining effect was blocked by a relatively selective M₃ antagonist 4-DAMP. Himbacine, an M₂/M₄ antagonist, or AFDX-116, a selective M₂ antagonist, completely blocked the inhibitory effect of oxotremorine-M on monosynaptic EPSCs. However, the specific M₄ antagonist MT-3 did not alter the effect of oxotremorine-M on monosynaptic EPSCs. Himbacine also partially attenuated the effect of oxotremorine-M on polysynaptic EPSCs in some cells, and this effect was abolished by 4-DAMP. Furthermore, oxotremorine-M significantly decreased spontaneous EPSCs in 7 of 22 (31.8%) neurons, an effect that was blocked by 4-DAMP. This study provides new information that the M₂ mAChRs play a critical role in the control of glutamatergic input from primary afferents to dorsal horn neurons. The M₃ and M₂/M₄ subtypes on a subpopulation of interneurons are important for regulation of glutamate release from interneurons in the spinal dorsal horn.