In a previous paper (1) to clarify the physiological significance of endogenous 5-hydroxytryptamine (5-HT) the subcellular distribution of 5-HT in rabbit brain stem has been studied and the electron microscopical structures of the fractions which contain 5-HT have been examined. 5-HT was principally found in P2-fraction which consisted largely of mitochondria and nerve-ending particles (NEPs). After density-gradient centrifugation 5-HT was found to be comparatively highly concentrated in NEPs-fraction (P2B-fraction). On the other hand suspension of P2-fraction in hypo-osmotic medium and subsequent differential centrifugation resulted in high concentration of 5-HT in synaptic vesicles (sv) fraction although considerable amount of 5-HT was also found in P2D- and P2S-fraction. In general 5-HT in brain stem was not so highly concentrated in specific fraction as with the distribution of acetylcholine (ACh) (2, 3) but rather diffusibly distributed among several fractions. In this paper the role of 5-HT in synaptic transmission, especially in central nervous system was investigated mainly through the experiments on 5-HT uptake by subcellular fraction of rabbit brain stem. The chemical transmission at synapses consists of the following consecutive steps: 1) release of transmitter by synaptic impulses. 2) combination of transmitter with postsynaptic receptor. 3) generation of impulses by postsynaptic potential. It appears that the study into the formation, uptake, storage, release and inactivation of 5-HT is necessary for full and accurate understanding of mechanism of chemical transmission, especially when there is no direct evidence for that 5-HT is synaptic transmitter in central nervous system. Segawa and Kuruma (4), Segawa et al. (5) in this laboratory have already shown that NEPs or sv of brain, when incubated in a medium containing 5-HT, could take up 5-HT from the medium and some drugs could inhibit the uptake of 5-HT. However we have as yet very little information as to whether NEPs or sv are specific uptake and storage site for 5-HT in situ. Also the mechanisms with which amine can be taken up by synapses and drug inhibits the uptake are poorly understood. The following is a systematic study concerning the mechanism of 5-HT uptake and drug effect.