The coupling of the muscarinic receptor to the inhibition of the adenylate cyclase system was studied in adult rat cortical tissue dissociated by teasing tissue minces through finely meshed Nitex filters. The intracellular ATP stores in the final preparation were metabolically prelabeled with [3H]adenine and the [3H]cAMP formed in the tissue was isolated by ion exchange chromatography. Forskolin (3-30 microM) elevated [3H]cAMP levels 5- to 9-fold over basal in the preparation, with maximum stimulation achieved by 10-15 min. In the dissociated cortex, carbachol inhibited forskolin-elevated [3H]cAMP levels with an EC50 value of 1.4 microM; maximal inhibition was in the range of 20-30%. Atropine completely blocked the response (Ki = 1.8 nM), which showed that carbachol stimulates a muscarinic receptor to inhibit [3H]cAMP levels in this preparation. Pirenzepine, an M1-selective antagonist, blocked the response to carbachol with low potency (Ki = 467 nM), which indicated that an M2 muscarinic receptor subtype mediates [3H]cAMP inhibition in the cortex. The response to 10 microM carbachol was not affected by 10 mM EGTA, 50 microM D-tubocurarine, or 100 nM tetrodotoxin; thus, activation of nicotinic receptors or a neuronal release process was not involved. [3H]cAMP reduction in response to muscarinic stimulation was also observed in dissociated tissue prepared from other brain regions. A robust response was encountered in striatal preparations (maximal inhibition 40%), while hippocampal responses were smaller and less reproducible than in the cortex. The striatal response was shown to be pharmacologically similar to the cortical response.