Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[3H]methylscopolamine [( 3H]NMS) binds to a single site in cultures with a KD of 89 pM and a Bmax of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [3H]NMS were performed to determine what receptor subtypes were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 (11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one), with Ki values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M1 subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [3H]NMS from two sites with Ki values of 6.5 and 147 microM, with the higher-affinity form predominant (83% of sites). The M1 receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC50 of 37 microM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.