The rate-limiting reaction in the biosynthesis of the neurotransmitter, serotonin, is catalyzed by the enzyme, tryptophan hydroxylase. Studies on the characteristics of this enzyme have been hampered by its relative instability and paucity in the brain. We have modified a charcoal adsorption radioenzymatic assay used for the measurement of tyrosine hydroxylase to assess rat brain tryptophan hydroxylase activity. This protocol is based on the principle that aromatic amino acid hydroxylases are mixed-function oxygenases and will utilize O2 and reduced pterin to convert tritiated amino acid substrate to product and tritiated H2O. All product and unreacted substrate are adsorbed by acidified charcoal. The [3H]H2O is analyzed by liquid scintillation spectrometry and is indicative (stoichiometrically) of the amount of product formed and, thus, the activity of the enzyme. This assay has a high signal-to-noise ratio and is sensitive enough to determine enzymatic activity in homogenates of individual raphe nuclei. In addition, its simplicity in design allows for the simultaneous testing of large numbers of samples. The enzyme activity and kinetic determinations derived from this protocol agree with those of other investigators using more lengthy, involved procedures.