There is considerable interest in the regulation of the extracellular compartment of the transmitter serotonin (5-hydroxytryptamine, 5-HT) in the midbrain raphe nuclei because it can control the activity of ascending serotonergic systems and the release of 5-HT in terminal areas of the forebrain. Several intrinsic and extrinsic factors of 5-HT neurons that regulate 5-HT release in the dorsal (DR) and median (MnR) raphe nucleus are reviewed in this article. Despite its high concentration in the extracellular space of the raphe nuclei, the origin of this pool of the transmitter remains to be determined. Regardless of its origin, is has been shown that the release of 5-HT in the rostral raphe nuclei is partly dependent on impulse flow and Ca(2+) ions. The release in the DR and MnR is critically dependent on the activation of 5-HT autoreceptors in these nuclei. Yet, it appears that 5-HT autoreceptors do not tonically inhibit 5-HT release in the raphe nuclei but rather play a role as sensors that respond to an excess of the endogenous transmitter. Both DR and MnR are equally responsive to the reduction of 5-HT release elicited by the local perfusion of 5-HT(1A) receptor agonists. In contrast, the effects of selective 5-HT(1B) receptor agonists are more pronounced in the MnR than in the DR. However, the cellular localization of 5-HT(1B) receptors in the raphe nuclei remains to be established. Furthermore, endogenous noradrenaline and GABA tonically regulate the extracellular concentration of 5-HT although the degree of tonicity appears to depend upon the sleep/wake cycle and the behavioral state of the animal. Glutamate exerts a phasic facilitatory control over the release of 5-HT in the raphe nuclei through ionotropic glutamate receptors. Overall, it appears that the extracellular concentration of 5-HT in the DR and the MnR is tightly controlled by intrinsic serotonergic mechanisms as well as afferent connections.