Acetylcholine released from the cerebral cortex was collected using microdialysis while stimulating the region of the pedunculopontine tegmentum in urethane-anesthetized rats. Electrical stimulation in the form of short trains of pulses delivered once per minute produced a 350% increase in acetylcholine release and a desynchronization of the electroencephalogram, as measured by relative power in the 20-45 Hz range (low-voltage fast activity). Perfusion of the region of cholinergic neurons believed to be responsible for the cortical release of acetylcholine, the nucleus basalis magnocellularis, was carried out using a second microdialysis probe. Exposure of the nucleus basalis magnocellularis to blockers of neural activity (tetrodotoxin or procaine) or to blockers of synaptic transmission (calcium-free solution plus magnesium or cobalt) produced a substantial decrease in the release of acetylcholine and desynchronization evoked by brainstem stimulation. Exposure of the nucleus basalis magnocellularis to the glutamate antagonist, kynurenate, resulted in a decrease in evoked acetylcholine release and electroencephalogram desynchronization similar in magnitude to that produced by nonspecific blockers, whereas application of muscarinic or nicotinic cholinergic blockers to the nucleus basalis magnocellularis did not reduce acetylcholine release or electroencephalogram desynchronization. Application of tetrodotoxin to the collection site in the cortex abolished the stimulation-evoked acetylcholine release, but not the low baseline release indicating that cholinergic nucleus basalis magnocellularis neurons have a low spontaneous firing rate in urethane-anesthetized animals. The results of this study suggest that the major excitatory input to the cholinergic neurons of the nucleus basalis magnocellularis from the pedunculopontine tegmentum is via glutamatergic and not cholinergic synapses.