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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.