In male rats, the effects of the administration of the novel serotonergic agent flibanserin on the synthesis of 5-HT were evaluated in the frontal cortex (FC), hippocampus (Hip) and brainstem (Br). The selective serotonergic uptake blocker, fluoxetine, and two serotonin1A (5-HT1A) agonists, 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) and buspirone, were used as reference compounds. The synthesis of 5-HT was assessed by measuring the accumulation of 5-hydroxytryptophan (5-HTP) after blockade of aromatic amino acid decarboxylase induced by m-hydroxybenzylhydrazine (NSD-1015), at 100 mg/kg i.p., 30 min before sacrifice. Flibanserin, 8-OH-DPAT and buspirone were given 15 min before NSD-1015, while fluoxetine 120 min before NSD-1015. In our experimental conditions, a different efficacy, expressed as percentage of maximal inhibition (Max) of 5-HTP accumulation, and a different potency, expressed in terms of minimal effective dose (MED), were observed in different brain areas with tested compounds. Flibanserin (1-32 mg/kg) decreased 5-HT synthesis with preferential activity in the FC, compared to the Hip and Br, both in terms of potency (MED=2 mg/kg in FC, 16 mg/kg in Hip and Br) and efficacy (Max=65% in FC, 44% in Hip and 29% in Br). Fluoxetine (1-30 mg/kg) decreased 5-HT synthesis with preferential activity in FC than in Hip and Br, only in terms of potency (MED=3 mg/kg in FC, 10 mg/kg in Hip and Br), this result being similar to that observed for flibanserin. In contrast, it showed greater efficacy both in FC and Hip (Max about 60%), than in Br (Max=49%). On the contrary, 8-OH-DPAT (0.3-3 mg/kg) decreased 5-HT synthesis with the same potency in all brain regions (MED=3 mg/kg) and showed the greatest efficacy in FC than in Hip and Br (Max=56% in FC, 49% in Hip and 40% in Br). Furthermore, buspirone (3-30 mg/kg), while inhibiting 5-HTP accumulation in all areas with the same efficacy (Max about 30%), seemed to have higher potency in Br than in FC and Hip (MED=3 mg/kg in Br, 10 mg/kg in FC and Hip). The results in terms of regional differences are discussed.