We measured pharmacologically isolated GABAergic currents from layer II/III neurons of the rat auditory cortex using patch-clamp recording. Activation of muscarinic receptors by muscarine (1 µM) or oxotremorine (10 µM) decreased the amplitude of electrically evoked inhibitory postsynaptic currents to about one third of their control value. Neither miniature nor exogenously-evoked GABAergic currents were altered by the presence of muscarinic agonists, indicating that the effect was spike-dependent and not mediated postsynaptically. The presence of the N- or P/Q- type Ca²⁺-channel blockers -Conotoxin GVIA (1 µM) or -AgaTx TK (200 nM) greatly blocked the muscarinic effect, suggesting that Ca²⁺-channels were target of the muscarinic modulation. The presence of the muscarinic M₂ receptor (M₂R) antagonists methoctramine (5 µM) or AF-DX 116 (1 µM) blocked most of the muscarinic eIPSC reduction, indicating that M2Rs were responsible for the effect, while the remaining component of the depression displayed M₁R-like sensitivity. Tissue pre-incubation with the specific blockers of phosphatidyl-inositol-3-kinase (PI₃K) wortmannin (200 nM), LY294002 (1 µM) or with the Ca²⁺-dependent PKC-inhibitor Go 6976 (200 nM), greatly impaired the muscarinic decrease of the eIPSC amplitude, while the remaining component was sensitive to preincubation in the phospholipase C blocker U73122 (10 µM). We conclude that acetylcholine release enhances the excitability of the auditory cortex by decreasing the release of GABA by inhibiting axonal V-dependent Ca²⁺-channels, mostly through activation of presynaptic M2Rs/PI3K/Ca²⁺-independent PKC pathway and -to a smaller extent- by the activation of M₁/PLC/Ca²⁺- dependent PKC.