It has been shown that ethanol produces a complex interoceptive cue in rodents with distinct GABAergic, glutamatergic, and serotonergic (5-hydroxytryptamine, 5-HT) components. The present study aimed to examine the contribution of the 5-HT system originating in the dorsal raphe nucleus (DRN) to the discriminative stimulus effects of ethanol in male Wistar rats. Therefore, selective lesions of 5-HT neurons in the DRN were induced by microinfusions of 5,7-dihydroxytryptamine. The DRN- and sham-lesioned rats were trained to discriminate ethanol (1.0 g/kg) from saline in a standard two-lever drug discrimination procedure. Acquisition of ethanol discrimination and discrimination performance after consumption of lower doses of ethanol did not differ between the groups. In substitution tests, diazepam (0.5-2.5 mg/kg), a nonselective benzodiazepine receptor agonist, partially generalized from the ethanol cue in both groups. In contrast, m-chlorophenylpiperazine (0.1-0.9 mg/kg), a mixed 5-HT(1B/2C) receptor agonist, did not mimic the ethanol cue. The drug decreased response rates in both groups, but this effect was more evident in the sham-lesioned group. A 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propyloamino)-tetraline (0.05-0.4 mg/kg), did not produce significant increase in ethanol-appropriate responding in either group. These results may indicate that 5-HT neurons of the DRN are not critically involved in ethanol discrimination in the rat.