The anxiolytic effect of ethanol is generally accepted to be involved in the development of alcohol dependence. Because serotonin (5-HT) is said to be involved in both anxiety and alcohol dependence, in the present study the effect of acute ethanol administration on basal 5-HT release of the medial prefrontal cortex and its effect on 5-HT release in rats submitted to an animal model of anxiety, the elevated plus maze test, were detected in two rat strains showing a different anxiety-related behavior. Ethanol had an anxiolytic-like effect and induced an increase of basal 5-HT release in the medial prefrontal cortex in the home cage in the less anxious Wistar–Harlan rats. Both effects were not seen in the more anxious Wistar–BgVV rats. The exposure to the elevated plus maze test induced an increase of extracellular 5-HT in the more anxious Wistar–BgVV rats but not in the less anxious Wistar–Harlan rats. Ethanol did not change 5-HT release during the elevated plus maze test in both rat strains. Thus, the anxiolytic-like effect of ethanol in Wistar–Harlan rats was not primarily associated with a decrease of 5-HT release in the prefrontal cortex as it is seen with other anxiolytic agents like diazepam.