This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 X 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 micrograms protein with 150 microM L-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37 degrees C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 micrograms hog kidney aromatic-L-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37 degrees C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37 degrees C may reach 14,000 dpm, whereas the blank only has 300 dpm (less than 3% of test value). This microassay is fast (less than 2 h to complete all reactions) and convenient for performing a large number of determinations.