We analyze the time course of 5-hydroxytryptamine (5-HT, serotonin) release from K+-depolarized hippocampal slices using a two-compartment kinetic model. The model is based on the assumptions that the rate of release is dependent on the amount of 5-HT in a releasable pool and that this pool may be resupplied during depolarization by newly synthesized 5-HT. Comparisons were made between predictions of the model and observed changes in 5-HT metabolism and in 5-HT release studied under a variety of experimental conditions. In accordance with predictions of the model, experimental manipulation of 5-HT synthesis and breakdown rates did not affect release immediately after depolarization but did affect the release rate during prolonged depolarization. Increasing bath tryptophan from 0 to 10 microM approximately doubled both 5-HT synthesis and the release rate after 40 min of K+-induced depolarization while having a smaller effect on release during the first 2 min. Inhibition of 5-HT breakdown did not significantly affect release during the first 2 min of depolarization but increased it over threefold after 40 min. In contrast, altering the concentrations of K+ or Ca2+ in the incubation medium affected mainly the early phase of 5-HT release and not the late phase. Reducing Ca2+ from 2.4 to 0.4 mM reduced 5-HT release by about 30% during the first 9 min of depolarization but did not affect release during the subsequent 30 min. Increasing the concentration of K+ from 18 to 60 mM stimulated release by sixfold during the first 2 min but only twofold after a subsequent 30 min. These results support our kinetic model and suggest that regulation of 5-HT metabolism at the site of the nerve terminal could be a mechanism for modulation of 5-HT release during prolonged discharge of serotonergic neurons.