Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.