Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 microM) alone did not affect the basal release of adenosine. However, carbachol (100 microM) induced a 253% increase in NMDA (20 microM)-evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 microM) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (1 microM). Neither an M1-selective dose of pirenzepine (50 nM) nor the M2-selective antagonist methoctramine (1 microM) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.