The substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-chloro-amphetamine (PCA) and fenfluramine (FEN) all exert their effects by releasing serotonin (5-HT) from presynaptic nerve terminals. In the current study, we examined the ability of these agents to induce the release of 5-HT in culture fetal raphe neurons. The data indicate that the rank order of release potencies for these agents was (+/-)PCA>(+)MDMA=(+)MDA=(+/-)FEN. Studies examining the role fo calcium in 5-HT release demonstrate that preventing calcium influx with L- and N-type calcium channel blockers inhibits potassium-stimulated release of -3H-5-HT but has no effect on release induced by the substituted amphetamines. Furthermore, omitting calcium from the extracellular media or depleting the vesicular pool of neurotransmitter with continual potassium stimulation did not affect the release of -3H-5-HT induced by these compounds. Administration of fluoxetine prior to the substituted amphetamines significantly attenuated the releasing effects of these agents, while producing no effect on potassium-stimulated release. These results are consistent with the notion that the amphetamines induce release of cytoplasmic 5-HT via the plasma membrane transporter.