In this study we investigated, using in vivo microdialysis and single unit recordings, the role of serotonin4 (5-HT4) receptors in the control of nigrostriatal and mesoaccumbal dopaminergic (DA) pathway activity. In freely moving rats, the 5-HT4 antagonist GR 125487 (1 mg/kg, i.p.), without effect on its own, significantly reduced the enhancement of striatal DA outflow induced by 0.01 (−35%) and 0.1 (−66%), but not 1 mg/kg, s.c. haloperidol (HAL). Intrastriatal infusion of GR 125487 (1 µm) had no influence on basal DA outflow, but attenuated (−49%) the effect of 0.01 mg/kg HAL. Systemic administration of GR 125487 modified neither basal nor 0.01 mg/kg HAL-stimulated accumbal DA outflow. In halothane-anaesthetized rats, 1 or 10 mg/kg GR 125487, without effect by itself, failed to modify the changes in accumbal and striatal DA outflow elicited by electrical stimulation (300 µA, 1 ms, 20 Hz, 15 min) of the dorsal raphe nucleus. Finally, GR 125487 (444 µg/kg, i.v.), whilst not affecting basal firing of DA neurons within either the substantia nigra or the ventral tegmental area, reduced HAL-stimulated (1–300 µg/kg, i.v.) impulse flow of nigrostriatal DA neurons only. These results indicate that 5-HT4 receptors exert a facilitatory control on both striatal DA release and nigral DA neuron impulse flow only when nigrostriatal DA transmission is under activated conditions. Furthermore, they indicate that the striatum constitutes a major site for the expression of the control exerted by 5-HT4 receptors on DA release. In contrast, 5-HT4 receptors have no influence on mesoaccumbal DA activity in either basal or activated conditions.