Regional concentrations of serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) were measured by high performance liquid chromatography in 16 discrete brain areas of non-stressed control Wistar rats and of stressed Wistar rats. Stress was induced by immobilization for one hour. Also studied were the effects of morphine (10 mg/kg, i.p.) and diazepam (5 mg/kg, i.p.) on concentrations of 5HT and 5HIAA in 10 discrete brain areas of the non-stressed as well as stressed rats. In control rats, regional concentrations of 5HT and 5HIAA varied in the different areas of the prefrontal cortices, limbic structures, striatum and hypothalamus with the highest 5HT contents in the area hypothalamicus lateralis and the lowest in the anterior cingulate cortex. Acute immobilization induced increases in 5HIAA content in prefrontal cortex polar field (FCP), prefrontal cortex medial field (FCM), n. preopticus medialis (PM), area hypothalamicus lateralis (L) and n. dorsomedialis (DM) and also increases in 5HT content in the PM, L, DM, and n. arcuatus median eminence (ARC-ME). In the non-stressed rats, morphine caused an elevation of 5HIAA concentration in the striatum and PM, and also an elevation of 5HT content in the PM and DM, while diazepam induced no alteration in the concentrations of 5HIAA and 5HT. In the stressed rats, morphine potentiated the stress-induced elevation of 5HIAA content in the striatum and attenuated the stress-induced elevation of 5HIAA in the PM and L, and that of 5HT in the PM, remaining 5HIAA content in FCP and FCM unaltered. Diazepam canceled the stress-induced increase in 5HIAA levels in FCP, FCM, CP and DM and in 5HT levels in DM. These results suggest that diazepam may reduce fear and anxiety by attenuating stress-induced increase of serotonin turnover.