In this study, we investigated the effects of putrescine, a precursor of polyamine metabolism, on depolarization (potassium chloride, KCl)-induced [³H]serotonin (5-hydroxytryptamine (5-HT) release from brain synaptosomes of rainbow trout (Oncorhynchus mykiss). An irreversible inhibitor of polyamine metabolism, difluoromethyl-ornithine (DFMO), inhibited the KCl-induced [³H]serotonin efflux, and addition of putrescine (PUT) reversed the inhibitory effects of the former in synaptosomal preparations. In Fura-2/AM loaded synaptosomes, KCl stimulated Ca²⁺ influx. Hence, prior addition of EGTA abolished the KCl-induced Ca²⁺ responses, indicating that KCl-induced depolarization recruited Ca²⁺ from extracellular medium. The DFMO curtailed the KCl-evoked Ca²⁺ influx, and addition of PUT reversed the inhibitory effects of DFMO. Furthermore, fish brain was found to contain polyamines in high amounts (e.g. putrescine from 600 ± 20 to 850 ± 30 nmol/g protein). These results suggest that polyamine putrescine, present in fish brain, might be involved in [³H]serotonin efflux via Ca²⁺ influx mechanisms.