Abstract The formation of 3H 2O from L-4- 3H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of 3H 2O from L-4- 3H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank 3H 2O produced from L-4- 3H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank 3H 2O produced from L-3,5- 3H-tyrosine. The K m values of tyrosine hydroxylase for phenylalanine determined by the production of 3H 2O from L-4- 3H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of 3H 2O formed from L-4- 3H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.