Release of endogenous serotonin [5-hydroxy-tryptamine (5-HT)] in the cerebellum of awake rats was characterized using in vivo microdialysis. 5-HT output was increased (approximately 70%) by local application of KCI (100 mM) and was reduced (approximately 60%) by both tetrodotoxin (0.5 microM) and omission of Ca2+ from the perfusion fluid. 5-HT release was decreased (approximately 70%) by the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (0.25 mg/kg, s.c.), and this effect was rapidly reversed by a selective 5-HT1A antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo- hexane-carboxamide trihydrochloride (WAY-100635; 0.1 mg/kg, i.p.). These results indicate that a large portion of the measurable 5-HT output in the cerebellum is of neuronal origin, is dependent on impulse flow, and is sensitive to 5-HT1A autoreceptor activation. Further studies examined the relationship between 5-HT levels and general activity of the animals across the light-dark transition and during behavioral manipulations. Both 5-HT levels and behavioral activity were significantly elevated during the dark period, with changes in 5-HT efflux closely paralleling changes in activity. Similar increases (approximately 40%) in 5-HT output were observed during both feeding and feeding in the presence of a stressor (tail pinch). These findings suggest that behavioral state is an important factor determining neuronal 5-HT release in cerebellum under physiological conditions.