Fast cyclic voltammetry (FCV) at carbon fibre microelectrodes was used to monitor the time course of changes in extracellular concentration of endogenous 5-hydroxytryptamine ([5-HT]ex), in slices of rat brain containing either dorsal raphe nucleus (DRN) or suprachiasmatic nucleus (SCN). Five- or 20-pulse electrical stimulation trains were applied at frequencies between 5 and 500 Hz. [5-HT]ex was frequency and train length dependent, with maximum overflow in both sites at 100 Hz. Methiothepin (0.1 microM) caused a significant increase in [5-HT]ex when 5 pulses were applied at 5 Hz or when 20 pulses were applied at 10 or 20 Hz, but not at higher frequencies. When a single pseudo single pulse stimulation was used (5-pulse train at 100 Hz), methiothepin (0.1 microM) did not enhance [5-HT]ex in either the DRN or SCN; when 4 such pseudo single pulses were applied at 1 Hz methiothepin (0.1 microM) enhanced [5-HT]ex. A minimum period of stimulation of between 400 ms and 1 s was required for autoreceptor activation to occur in both regions. We conclude that [5-HT]ex can show a wide dynamic range of response to electrical stimulation, and that in both DRN and SCN it is subject to pulse-to-pulse regulation by presynaptic autoreceptors.