The distribution of 5-hydroxytryptamine (HT) was studied in various cytoplasmic fractions of rat brain and 75% was found to be present in the mitochondrial fraction, with the remainder largely in the nuclear and supernatant fractions. A number of inhibitors of oxidative phosphorylation were tested for their effect on HT binding by mitochondria. Dinitrophenol did not affect binding, while phenyl ether, dibenamine, reserpine, and LSD were potent inhibitors. Although reserpine brought about complete HT release from mitochondria, the effect was immediately reversible after washing. The significance of these results in terms of mitochondrial receptor sites is discussed.