Previous investigations have demonstrated that compounds capable of blocking presynaptic potassium channels can stimulate neurotransmitter release at both peripheral and central synapses. This study examined the in vitro effects of the "classical" potassium channel blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the spontaneous basal release of [3H]5-hydroxytryptamine ([3H]5-HT) from rat hippocampal slices using an automated superfusion apparatus. 4-AP and structural analogs increased the spontaneous basal release of [3H]5-HT in a concentration-related manner. The rank order of potencies from the estimated EC50 values indicated that 3,4-diaminopyridine (0.88 mM) approximately 4-AP (1.2 mM) > 2-AP (89 mM) > 3-AP (100 mM) > pyridine (256 mM). TEA stimulated [3H]5-HT release with an estimated EC50 value of 63 mM and was less efficacious than the pyridine congeners. The enhancement of release induced by 1 mM 4-AP was additive with 100 mM TEA and 5 microM veratridine but not with 3,4-diaminopyridine or KCl (25 and 50 mM). The release induced by 4-AP (0.3, 1 and 10 mM) and TEA (30, 100 and 300 mM) was significantly attenuated in a calcium-free buffer containing 1 mM ethylene glycol-bis(b-aminoethyl ether N,N,N',N'-tetraacetic acid. Tetrodotoxin (1 microM), a sodium channel blocker, was unable to block the response to 4-AP (1 mM) and TEA (100 mM). Notably, this concentration of tetrodotoxin reduced the stimulation of [3H]5-HT release produced by the sodium channel opener veratridine (5 microM). Taken together, the results demonstrate that potassium channel blockade can enhance the spontaneous basal release of [3H]5-HT in rat hippocampal slices. These effects are at least partly dependent on extracellular calcium and do not appear to be mediated by modulating sodium channel function.