It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (R-N) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic R-N increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M-1,M-4 muscarinic receptor antagonist pirenzepine but not by M-2 or M-3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M-2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing R-N and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M-2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M-1- and M-2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.