Fast scan cyclic voltammetry with carbon fiber electrodes has been used to investigate the dynamics of the neurotransmitter 5-hydroxytryptamine (5-HT) in the extracellular fluid of two brain regions: the dorsal raphe and the substantia nigra reticulata. The method used previously was shown to be optimized to allow the time course of 5-HT concentration changes to be measured rapidly. Measurements were made in slices prepared from the brains of rats with the carbon fiber electrode inserted into the tissue and a bipolar stimulating electrode placed on the slice surface. Identification of 5-HT as the detected substance in both regions was based on voltammetric, anatomical, physiological, and pharmacological evidence. Autoradiography using [3H]paroxetine revealed highest 5-HT transporter binding densities in the regions in which voltammetric measurements were made. Evaluation of the pharmacological actions of tetrodotoxin and tetrabenazine, as well as the effects of calcium removal, suggested that 5-HT storage was vesicular and that the release process was exocytotic. The effects of fluoxetine (0.5 microM) were typical of a competitive uptake inhibitor, changing Km with little effect on Vmax. Release of 5-HT was found to be maximal with wide (2-ms) stimulus pulses in both regions, as expected for release from small unmyelinated processes, and to increase linearly with the number of pulses when high frequencies (100 Hz) were used. At lower frequencies, the concentration observed was a function of both release and uptake. Kinetic simulations of the data revealed that the major difference in 5-HT neurotransmission between the two regions was that release and uptake rates are twice as large in the dorsal raphe ([5-HT] per pulse = 100 +/- 20 nM, Vmax = 1,300 +/- 20 nM/s for dorsal raphe; [5-HT] per pulse = 55 +/- 7 nM, Vmax = 570 +/- 70 nM/s for substantia nigra reticulata). When normalized to tissue content, uptake rates in both regions were identical and similar to rates previously reported for dopamine in dopamine terminal regions. Nonetheless, compared with dopaminergic transmission in terminal regions such as the striatum, the absolute clearance rates in the substantia nigra reticulata and dorsal raphe were lower, resulting in a longer lifetime of 5-HT in the extracellular fluid and allowing long-range interactions.