Flibanserin, a 5-HT(1A) receptor agonist and 5-HT(2A) receptor antagonist, is being developed for the treatment of hypoactive sexual desire disorder (HSDD) in pre-menopausal women. Here, we investigated the effects of acute administration of flibanserin (15 and 45 mg/kg, p.o.) and the selective 5-HT(1A) receptor agonist (+)-8-OH-DPAT (1 mg/kg, i.p.) on neurotransmitter levels in brain areas of female rats. Specifically, levels of dopamine (DA) and serotonin (5-HT) and neurotransmitter metabolites were examined in prefrontal cortex (PFC), nucleus accumbens, hypothalamus and brain stem using high performance liquid chromatography coupled to electrochemical detection. In addition, spontaneous motor activity was determined in an automated motor activity system. Flibanserin (45 mg/kg) but not (+)-8-OH-DPAT significantly reduced motor activity, when compared to vehicle controls. Specifically, the DA turnover was significantly increased (279%) in the PFC after flibanserin treatment but less pronounced (159%) after 8-OH-DPAT administration. Serotonin tissue levels were not altered in any of the investigated brain regions upon flibanserin treatment. However, flibanserin produced a significant decrease of the major serotonin metabolite 5-hydroxyindoleacetic acid and 5-HT turnover in the PFC, nucleus accumbens, hypothalamus and brain stem similar to (+)-8-OH-DPAT. In conclusion, the present study indicates that flibanserin is able to modulate dopaminergic and serotonergic activity in distinct brain areas. The observed effects in the PFC on dopaminergic markers are different from those induced by (+)-8-OH-DPAT and may contribute to its therapeutic efficacy in HSDD. The effects of flibanserin on spontaneous motor behaviour are in agreement with its receptor profile and underscore that flibanserin is devoid of any locomotor hyperactivity inducing properties.