Abstract Since serotonin (5-HT) reportedly is involved in aberrant drinking of ethyl alcohol, the present study examined a possible role of the concentration of 5-HT in systems originating in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN) or both nuclei. The preference for alcohol offered in concentrations increased over 10 days from, 3% to 30% was determined for each Sprague-Dawley rat. After the rats were anesthetized with sodium pentobarbital, either 10 μg 5,7-DHT or artificial cerebrospinal fluid (CSF) was micro-injected stereotaxically into the DRN, MRN or both nuclei. After 10 days, a second alcohol preference test was offered to the animals. Then the rats were decapitated, each brain removed and the block of tissue containing injection sites was saved for histological analysis. The remaining portion was dissected into separate regions for analysis by HPLC of 5-HT, 5-hydroxyindoleacetic-acid (5-HIAA), norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The 5,7-DHT lesion of the DRN depleted the levels of 5-HT and 5-HIAA by 50–55% in the midbrain and pons and by 70–80% in the frontal cortex, whereas, the 5,7-DHT lesion of MRN reduced 5-HT in all regions except the corpus striatum. The depletion of 5-HT was lower in MRN-lesioned than in DRN-lesioned rats in the frontal cortex and nucleus accumbens. The combined lesion of both DRN and MRN produced a massive decline of > 90% of 5-HT and 5-HIAA in all structures except the pons where 5-HT was reduced by 70%. Whereas the level of NE was reduced mainly in the frontal cortex, the levels of DA and its metabolites were essentially unaffected by the 5,7-DHT lesions. Although single or combined lesions of the DRN and MRN failed to alter the intake of alcohol of the rats, the combined serotonergic lesions increased significantly the ingestion of water but not food. Correlational analyses in the sham groups showed a negative association between the intake of alcohol and cortical dopamine and possibly hippocampal 5-HT and NE as well as between the ingestion of food and 5-HT in the frontal cortex. Taken together, these observations in the Sprague-Dawley rat suggest that lower levels of these monoamines in certain regions of the brain may play a role in the maintenance of the basal intake of alcohol but not in the drinking after the injection of 5,7-DHT. Explanations of our findings include: (1) a compensatory neurochemical change in pre- or postsynaptic 5-HT receptors subsequent to the dysfunction of serotonergic neurons in the forebrain; (2) a unique characteristic of the Sprague-Dawley strain of rat; and (3) residual quanta of 5-HT which sustains the pattern of alcohol drinking.