The aim of the present study was to investigate the role of different endogenous opioid systems in the expression of ethanol's discriminative stimulus effects in a two-lever operant drug discrimination paradigm. Wistar rats were trained to make differential responses following the administration of ethanol (1 g/kg, IP) or saline. The correct response (fixed-ratio schedule; FR10) resulted in the presentation of food. Once rats had acquired the discrimination an ethanol dose-response test was conducted. The effects of opioid antagonists on the discrimination were assessed by administering the μ-opioid receptor antagonists naloxone (0.5–20 mg/kg SC) and cyprodime (5–100 mg/kg SC) and the δ-opioid receptor antagonist naltrindole (0.1–25 mg/kg SC) 15–30 min before the discrimination test. Furthermore, the selective κ-opioid antagonist nor-binaltorphimine (5 mg/kg SC) given 24 h before the test session was examined. Results of generalization testing demonstrate that ethanol discrimination was dose dependent. Pretreatment with naloxone produced only at the highest dose a partial, but significant, antagonism, whereas cyprodime failed to alter the ethanol cue. This suggested the involvement of other opioid receptor subtypes. However, neither naltrindole nor nor-binaltorphimine had any effect on the ethanol-saline discrimination. These results demonstrate that the expression of the ethanol cue is only partly dependent on the function of endogenous opioid systems.