Acetylcholine (ACh) in the medial pontine reticular formation (mPRF) originates from the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei and contributes to generating rapid eye movement (REM) sleep. The mechanisms controlling mPRF ACh levels are incompletely understood. This study tested the hypothesis that mPRF ACh release is regulated, in part, by muscarinic autoreceptors. The mPRF of intact, halothane-anesthetized cats was dialyzed with Ringer's solution (control) or Ringer's containing the muscarinic antagonist scopolamine, Scopolamine caused a dose-dependent increase in mPRF ACh release and a concomitant decrease in the number of halothane-induced cortical EEG spindles. These data suggest that presynaptic muscarinic receptors, presumed to reside on cholinergic LDT/PPT terminals in the mPRF, play a role in regulating mPRF ACh release, REM sleep and EEG spindles.