The serotoninergic (5-HT) input from the dorsal raphe nucleus (DRN) to midbrain dopamine (DA) neurons is one of the most prominent. In this study, using standard extracellular single cell recording techniques we investigated the effects of electrical stimulation of the DRN on the spontaneous activity of substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) DA neurons in anesthetized rats. Poststimulus time histograms (PSTH) revealed two different types of response in both SNpc and VTA. Some cells exhibited an inhibition-excitation response while in other DA neurons the initial response was an excitation followed by an inhibition. In SNpc, 56% of the DA cells recorded were initially inhibited and 31% of the DA cells were initially excited. In contrast, 63% of VTA DA cells were initially excited and 34% were initially inhibited. Depletion of endogenous 5-HT by the neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), and the 5-HT synthesis inhibitor para-chlorophenylalanine (PCPA), almost completely eliminated the inhibition-excitation response in both SNpc and VTA DA cells, without changing the percentage of DA cells initially excited. Consequently, the proportion of DA neurons that were not affected by DR stimulation increased after 5-HT depletion (from 13% to 60% in SNpc and from 6% to 31% in VTA). In several DA cells, DRN stimulation caused important changes in firing rate and firing pattern. These data strongly suggest that the 5-HT input from the DRN is mainly inhibitory. It also suggests that 5-HT afferences modulate SNpc and VTA DA neurons in an opposite manner. Our results also suggest that non-5-HT inputs from DR can also modulate mesencephalic DA neurons. A differential modulation of VTA and SNpc DA neurons by 5-HT afferences from the DRN could have important implications for the development of drugs to treat schizophrenia or other neurologic and psychiatric diseases in which DA neurons are involved.