Cortico-thalamic glutamatergic afferents control neuronal activity in the neostriatum. Cholinergic interneurons modulate the activity of medium spiny neurons through both pre- and post-synaptic actions via the activation of muscarinic receptors. The muscarinic pre-synaptic modulation was analyzed electrophysiologically. The transmitter release, induced by 4-AP, was studied and the block of paired pulse facilitation (PPF) by different muscarinic receptor antagonists was analyzed. The GABA A antagonist bicuculline isolated the glutamatergic transmission. Muscarinic agonists decreased the frequency of random synaptic potentials induced by 4-AP in about 60% of the cases without changes in input resistance (R N ) of the post-synaptic neuron or in the mean amplitude of the synaptic events; indicating a presynaptic action. The administration of both 1 μM carbachol or 20 nM muscarine increased PPF. Muscarinic receptor antagonists blocked this action with a potency order: 3-α-chloroimperialine>4-DAMP>AFDX-116=<gallamine pirenzepine. The IC 5 0 's for the first three antagonists were (nM): 0.65, 1.1, and 3.0. Their respective Hill coefficients were: 1.9, 1.4, and 1.3. 3-α-Chloroimperialine reduced the PPF almost completely. The M 3 and the M 2 muscarinic receptor antagonists 4-DAMP and AFDX-116, given at saturating concentrations, consistently blocked only a part of the PPF but had additive effects when given together. These data are consistent with the existence of both M 2 and M 3 muscarinic receptors in striatal glutamatergic afferents.