For direct measurement of the extracellular concentration of serotonin (5-HT) in the dorsal raphe nucleus (DRN) over the sleep-wake cycle we used the technique of in vivo microdialysis in six freely moving, naturally sleeping cats whose behavioral state was polygraphically determined. Perfusate samples from microdialysis probes histologically localized to the DRN showed the following significantly different levels of extracellular 5-HT, with the numbers in parentheses indicating successively the mean value in fmol/5 microliters perfusate sample, the % level relative to waking, and the sample n: waking (4.02, 100%, n = 38) > slow wave sleep (2.02, 50%, n = 30) > REM sleep (1.61, 38%, n = 17). These data, to our knowledge the first direct DRN 5-HT measurements during behavioral state changes, directly parallel the levels of serotonergic neuronal action potential activity and suggest that DRN extracellular 5-HT is determined by this action potential activity through synaptic release by recurrent axonal collaterals in the DRN.