Abstract— A modified tritium release assay for the measurement of synaptosomal tyrosine hydroxyl-ation. with a sensitivity suitable for use on areas of the rat brain with a low density of catecholamine terminals. is described. The apparent Km, for tyrosine hydroxylase in the hippocampus was 9.3 μM. in the hypothalamus 6.1 μM and in the striatum 9.9 μM Preparations from all three regions showed a pH optimum of 6.0–6.2, and the activities were reduced to a small % of control by synaptosomal disruption. 3-iodotyrosine. noradrenaline and reserpine. Membrane depolarization at a pH of 6.1 did not elevate tyrosine hydroxylation rates in any of the regions studied, although striatal tyrosine hy-droxylation rates were elevated at a pH of 7.2 by 55 mM-K+. The addition of dibutyryl cyclic AMP (0.5 mM) to the medium produced a 20-30% elevation of the rates of hydroxylation in all three regions studied: addition of tetrahydrobiopterin (0.2 mM) elevated hydroxylation rates in the hypothalamus and striatum. These results indicate that many characteristics of tyrosine hydroxylase from the three regions are similar. In each case the enzyme is apparently sensitive to end-product inhibition and to cyclic AMP activation.