High level of positive affectivity acts as a protective factor against adverse effects of stress and decreases vulnerability to mood disorders and drug abuse. Fifty-kHz ultrasonic vocalizations (50-kHz USV) index the level of positive affect in the rat, whereas stable, trait-like inter-individual differences in terms of vocalization activity exist. Previously we have demonstrated that chronic stress can alter the effect of repeated amphetamine administration on 50-kHz vocalizations, and this effect is different in rats with high and low positive affectivity. In the present study it was tested whether the chronic stress effect on amphetamine-induced 50-kHz USV activity is altered by inhibition of serotonin reuptake. Male Wistar high (HC) and low (LC) 50-kHz vocalizing rats were subjected to 43-day chronic variable stress (CVS) regimen. On day 17 of the CVS, the four-week once a day fluoxetine (10 mg/kg) treatment was started. After the CVS and fluoxetine treatment, amphetamine (1 mg/kg) was daily administered for ten days and again nine days after withdrawal. Chronically stressed rats developed cross-sensitization of 50-kHz USV-s with repeated administration of amphetamine except the stressed LC rats that had not received fluoxetine. Amphetamine treatment decreased serotonin turnover in the fluoxetine-treated HC rats, but increased it in fluoxetine-treated LC rats. The effect of amphetamine on levels of amino acids in frontal cortex and hippocampus also depended on previous experience with chronic stress, repeated treatment with fluoxetine, and positive affectivity. Hence, this study provides further evidence the effects of chronic stress, psychostimulants, and a selective serotonin reuptake inhibitor are influenced by the inherent positive affectivity.