Cholinergic neurons in the pons appear to play a major role in generating rapid eye movement (REM) sleep. In the present study, acetylcholine and glutamate release in the pedunculopontine tegmental nucleus (PPT) during the sleep-waking cycle were investigated by in vivo microdialysis. Acetylcholine release during slow wave sleep (SWS) was significantly lower (P<0.05) than during REM sleep and wakefulness. On the other hand, glutamate release during wakefulness was higher (P<0.05) than during REM sleep and SWS. Furthermore, the application of N-methyl-D-aspartate (1 mM) induced a significant increase of nitric oxides (NOx) for 20 min (P<0.05) and a decrease of norepinephrine for the first 15 min (P=0.01), indicating NOx regulation on norepinephrine release in PPT.