This study tested a behavior-suppressing punishment system and how its activity may be altered by agents known to interrupt or enhance serotonergic (5-HT) transmission. Holtzman male albino rats were tested for shuttle box avoidance acquisition and intertrial responding either 1 or 8 h following daily injection of fenfluramine (FEN) or fluoxetine (FXT). When the drug-test interval was 1 h, a time when both drugs are presumably potentiating 5-HT activity, avoidance acquisition and intertrial responding were impaired. When testing occurred 8 h after drug treatment, a time when 5-HT level are unaltered by FXT and are maximally reduced by FEN, these drugs had no effect on avoidance acquisition, but FEN produced an increase in intertrial responses whereas FXT did not. These results support the proposal of an inhibitory 5-HT system. Furthermore, these data demonstrate that FEN is capable of exerting a biphasic action on intertrial responding and suggest that the time interval between drug administration and behavioral testing is a crucial variable when investigating FEN.