The histamine containing posterior hypothalamic region (PH-TMN) plays a key role in sleep-wake regulation. We investigated rapid changes in glutamate release in the PH-TMN across the sleep-wake cycle with a glutamate biosensor that allows the measurement of glutamate levels at 1-4 sec resolution. In the PH-TMN, glutamate levels increased in active waking (AW) and REM sleep compared to quiet waking and NREM sleep. There was a rapid (0.6 ± 1.8 sec) and progressive increase in glutamate levels at REM sleep onset. A reduction in glutamate levels consistently preceded the offset of REM sleep by 8 ± 3 sec. Short duration sleep deprivation resulted in a progressive increase in glutamate levels in the PH-TMN, perifornical-lateral hypothalamus (PF-LH) and cortex. We found that in the PF-LH, glutamate levels took a longer time to return to basal values compared to the time it took for glutamate levels to increase to peak values during AW onset. This is in contrast to other regions we studied in which the return to baseline values after AW was quicker than their rise with waking onset. In summary, we demonstrated an increase in glutamate levels in the PH-TMN with REM/AW onset and a drop in glutamate levels prior to the offset of REM. High temporal resolution measurement of glutamate levels reveals dynamic changes in release linked to the initiation and termination of REM sleep.