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Publisher Summary The enzyme tryptophan-5-hydroxylase is present in brain and several groups have been reported to be present in a crude mitochondrial fraction. Depending upon the mode of homogenization and the concentration of the original homogenate, about 40–50% of the tryptophan-5-hydroxylase activity can be obtained from the crude mitochondrial fraction. Equilibrium centrifugation of the crude mitochondrial fraction on a discontinuous sucrose gradient reveals that the tryptophan-5-hydroxylase activity is highest in the synaptosome fraction and lowest in the mitochondrial fraction. Therefore, it appears that the synaptosome contains tryptophan-5-hydroxylase, and the activity seen in the high speed supernatant fraction reflects the activity originally present in the cytoplasm of the nerve cell body where 5-HT is detected in low concentrations by fluorescence microscopy. The inhibitory effect of phenylalanine upon 5-HTP production from tryptophan in an isoosmotic homogenate of rabbit hindbrain is illustrated with a graph in this chapter. The chapter establishes that tryptophan is taken up by the synaptosome component of crude mitochondrial fractions. It then investigates the effect of phenylalanine upon this uptake. The physiological importance of tryptophan transport across the nerve cell membrane as a factor controlling the substrate concentration for tryptophan-5-hydroxylase is yet to be determined.