The rate of synthesis of serotonin (5-HT) in rat brain and spinal cord was determined by inhibiting monoamine oxidase and measuring the resulting accumulation of 5-HT. The location of this 5-HT was examined using fluorescence histochemistry. No alteration of accumulation was seen when the flow of impulses in serotonergic nerve fibers in the spinal cord was decreased by sectioning. The accumulation was also unaltered when the animals were treated with several drugs that reduce 5-HT turnover: LSD, psilocybin and chlorimipramine. These results support previous suggestions that normal control of synthesis of 5-HT by end-product inhibition or impulse-controlled feedback does not function in rats treated with a MAO inhibitor. The accumulation of 5-HT in brain was reduced when the animals were treated with N,N-dimethyltryptamine, α-ethyltryptamine, p-methoxy amphetamine or p-chloromethamphetamine after receiving a monoamine oxidase inhibitor. The same effect also occurred in both intact and transected spinal cords. These drugs also caused the appearance of extraneuronal 5-HT fluorescence. These effects probably mainly arose from release by these latter drugs of 5-HT which was not stored in granules. p-Chloromethamphetamine was also seen to deplete 5-HT from the granular stores of normal animals. p-Methoxyamphetamine, p-chloromethamphetamme and α-ethyltryptamine strongly potentiated the hindlimb extensor reflex in spinal rats whose endogenous 5-HT stores had been depleted. These drugs seem to directly stimulate the central 5-HT receptors involved in this reflex.