The signaling pathways mediating the muscarinic modulation of Ca2+ currents in neostriatal cholinergic interneurons were studied by combined patch-clamp recording and single-cell reverse transcription- PCR. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. These neurons expressed Q-, N-, L-, P-, and R-type Ca2+ currents and the mRNA for the alpha1 subunits believed to form the channels underlying these currents (classes A, B, C, D, and E). Of the interneurons tested, nearly all expressed M2-class (m2, m4) receptor mRNAs, whereas m1 receptor mRNA was found in only a subset (approximately 30%) of the sample. The muscarinic agonist oxotremorine methiodide produced a dose-dependent reduction of N- and P-type Ba2+ currents through Ca2+ channels that was antagonized by atropine. N- ethylmaleimide eliminated the modulation, as did preincubation with pertussis toxin. The onset and offset of the modulation were rapid and dose-dependent. The modulation was also attenuated by strong depolarizing prepulses and was not observed in cell-attached membrane patches. Taken together, our results suggest that activation of M2- class muscarinic receptors in cholinergic interneurons reduces N- and P- type Ca2+ currents through a membrane-delimited pathway using a Gi/o- class G-protein. This signaling pathway provides a cellular mechanism for hetero- and homosynaptic control of interneuronal activity and acetylcholine release in the striatum.