Systemic administration of selective 5-HT1A agonists, such as 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT), stimulates the electrical activity of ventral tegmental area (VTA) dopamine neurons by a mechanism which remains unknown. We have examined if this activation is dependent on glutamatergic, serotonergic and GABAergic neurotransmission and if 5-HT1A receptors located within the VTA or within the prefrontal cortex (PFC) could contribute. In vivo electrophysiological recordings were obtained from VTA dopamine neurons from anesthetized rats. The i.v. administration of the 5-HT1A agonist 8-OHDPAT induced a strong stimulation of burst and firing activity of dopamine neurons. This activation remained unchanged in rats pre-treated with the 5-HT depleting agent parachlorophenylalanine. However, pre-administration of the GABAB receptor antagonist phaclophen, but not of the GABAA antagonist picrotoxin, significantly reduced the 8-OHDPAT-induced activation. The N-methyl-d-aspartate (NMDA) antagonist MK 801 (dizocilpine), but not the AMPA/kainate antagonist [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)quinoxalin-1-yl] methyl-phosphonate (ZK 200775), partially prevented or reversed the effects of 8-OHDPAT. However, only the combined pre-administration of the two glutamate antagonists did completely prevent the activatory response to 8-OHDPAT and even converted the effect of 8-OHDPAT into an inhibition, in half of the dopamine neurons tested. Inactivation of the local 5-HT1A receptors by the microinfusion within the VTA of the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100,635), or of pertussis toxin, reduced the ability of 8-OHDPAT to stimulate the firing of dopamine neurons but not their burst activity. On the other hand, burst activation elicited by 8-OHDPAT was strongly reduced following the inactivation of prefrontal 5-HT1A receptors achieved by the microinfusion of WAY 100,635 within the PFC. These results show that activation of midbrain dopamine neurons by the systemic administration of 5-HT1A agonists does involve the inactivation of a tonic GABAergic tone, involving mainly the GABAB receptors, probably leading to the stimulation of a glutamatergic excitatory drive from the PFC to the VTA and an increase in glutamate release. This will excite dopamine neurons, preferentially through NMDA receptors. Furthermore, our results suggest that some 5-HT1A receptors located within the VTA may also participate in this activation.