The acute effects of two doses (0.5 and 1 mg/kg) of melatonin on the levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid in several rat brain regions were studied. Tryptophan content in the brain regions was unchanged by the treatments. Melatonin at a dosage of 0.5 mg/kg increased medial hypothalamic serotonin levels at 60 and 90 min after the injection. However, the dose of 1 mg/kg increased the levels of this amine or its metabolite in the preoptic area-anterior hypothalamus, medial and posterior hypothalamus, amygdala, and midbrain. These results suggest a specific regional sensitivity to melatonin as well as a dose-dependent response. The stimulatory melatonin effect on the serotoninergic system was also observed after a daily treatment with this hormone (0.5 mg/kg, twice daily during 10 days) in both intact or pinealectomized rats. In intact rats, melatonin treatment increased the levels of 5-hydroxyindole-3-acetic acid in the preoptic area-anterior hypothalamus and medial hypothalamus, while in pinealectomized rats melatonin increased the serotonin content in the medial hypothalamic region. The data support the idea that melatonin has a selective action on serotonin metabolism in regions that contain serotoninergic terminals, especially at medial hypothalamic level.