Simultaneous recording of complex spikes from multiple Purkinje cells (up to 44) in the rat cerebellum was used to examine the effects of 5-hydroxytryptamine (serotonin, 5-HT) on olivocerebellar function. Microinjection into the inferior olive was found to increase the average firing rate of inferior olivary neurons while slowing their oscillation frequency and increasing the coherence of their oscillations. Indeed, while the normal rostrocaudal band of synchronous activity remained unchanged, the degree of synchrony between Purkinje cell complex spikes within this band was enhanced following the 5-HT injections. Multiple-electrode recordings obtained from crus Ila and vermal lobule Vlb yielded qualitatively similar results; however, the effects on vermal activity were more pronounced. The effects of the 5-HT microinjection decayed with a time course of 75 min. The half-maximum effective concentration of 5-HT was between 10 and 100 microM. Injections of various 5-HT agonists and antagonists demonstrated that a 5-HT type-2A (5-HT2A) receptor is the main mediator for the 5-HT effect, which was very similar to the effect produced by injections of harmaline. However, 5-HT and harmaline appear to have independent mechanisms since the action of harmaline was not blocked by the 5-HT2A antagonist LY53857. A possible role for 5-HT, as a physiological enhancer of the timing of motor function of the olivocerebellar system, is discussed.