Previously, we reported that activation of muscarinic receptors modulates N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in auditory neocortex [Aramakis et al. (1997a) Exp Brain Res 113:484-496]. Here, we describe the muscarinic subtypes responsible for these modulatory effects, and a role for G-proteins and intracellular messengers. The muscarinic agonist oxotremorine-M (oxo-M), at 25-100 microM, produced a long-lasting enhancement of NMDA-induced membrane depolarizations. We examined the postsynaptic G-protein dependence of the modulatory effects of oxo-M with the use of the G-protein activator GTP gamma S and the nonhydrolyzable GDP analog GDP beta S. Intracellular infusion of GTP gamma S mimicked the facilitating actions of oxo-M. After obtaining the whole-cell recording configuration, there was a gradual, time-dependent increase of the NMDA receptor-mediated slow-EPSP, and of iontophoretic NMDA-induced membrane depolarizations. In contrast, intracellular infusion of either GDP beta S or the IP3 receptor antagonist heparin prevented oxo-M mediated enhancement of NMDA depolarizations. The muscarinic receptor involved in enhancement of NMDA iontophoretic responses is likely the M1 receptor, because the increase was prevented by pirenzepine, but not the M2 antagonists methoctramine or AF-DX 116. Oxo-M also reduced the amplitude of the pharmacologically isolated slow-EPSP, and this effect was blocked by M2 antagonists. Thus, muscarinic-mediated enhancement of NMDA responses involves activation of M1 receptors, leading to the engagement of a postsynaptic G-protein and subsequent IP3 receptor activity.