Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K(+)-induced [3H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations greater than 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K+ used to depolarize the synaptosomes and the concentration of external Ca2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [3H]5-HT release induced by the Ca(2+)-ionophore A 23187 or Ca(2+)-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K(+)-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca2+ channels and Ca2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [3H]nitrendipine to the dihydropyridine-sensitive L-type Ca2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K(+)-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K(+)-induced increase in intracellular free Ca2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K(+)-induced [3H]5-HT release by antagonizing voltage-dependent Ca2+ entry into nerve terminals.