High-speed centrifugation of preparations of brain (rat) has permitted the separation of two forms of 5-hydroxytryptamine (5-HT; serotonin), “bound” and “free”, and the influence of drugs on these forms has been studied. Normally, about 30 per cent of the total endogenous 5-HT is found in the free form. Some drugs known to depress or tranquilize (such as reserpine, chlorpromazine, phenobarbital), irrespective of their effect on total cerebral 5-HT, produce significant increases in the proportion of free 5-HT. The hallucinogenic agent, LSD-25, and the anti-depressant drugs imipramine and β-phenyl isopropylhydrazine lead to an elevation in total 5-HT, which may be accounted for by greater increases in bound than free 5-HT. The anti-depressant drug, iproniazid, produces a rise in both forms of the amine with no significant change in distribution. Among the anti-depressants, only iproniazid completely prevents the release of granule-bound 5-HT which ordinarily is induced by reserpine. This action is not entirely related to inhibition of monoamine oxidase or to the high levels of 5-HT produced by iproniazid.