In rats trained in an operant fixed-interval-30-s schedule of food reward (FI-30s), acute exposure to contingent footshock resulted in a response suppression that was released by diazepam (DZP; 4 mg/kg ip) but not by buspirone (0.25 or 0.50 mg/kg ip), Compared with baseline, hippocampal and cortical extracellular levels of serotonin (5-HText) did not change, regardless of operant period (punished or nonpunished) and drug. In contrast, in the striatum, an increase of 5-HText levels (535%) occurred during the punished period, counteracted by DZP, This effect was observed only in rats that were low responders during both nonpunished and punished periods, that is, those that exerted an efficacious control over responding. Uncontrollable shocks or exposure to an unfamiliar open field did not modify striatal 5-HText. Together, these results suggest that an acute activation of 5-HT neurons afferent to the striatum allows the rats to efficiently block responses that are negatively reinforced.