Prostaglandin H synthase catalyzes the formation of prostaglandin (PG) G2 from arachidonic acid (cyclooxygenase activity), and also the reduction of PGG2 to PGH2 (peroxidase activity). The ability of the pure synthase to accumulate the hydroperoxide, PGG2, under conditions allowing the concurrent function of both catalytic activities was investigated. The peroxidase velocity was continuously determined from the absorbance increases at 611 nm that accompanied oxidation of a peroxidase cosubstrate, N,N,N',N'-tetramethylphenylenediamine, and PGG2 concentrations were calculated from the peroxidase velocities and the peroxidase Vmax and Km values. Cyclooxygenase velocities were then calculated from the changes in PGG2. Parallel reactions monitored by the use of radiolabelled arachidonate or with a polarographic oxygen electrode were used to confirm the calculated PGG2 levels and the cyclooxygenase velocities. The concentration of PGG2 was found to follow a transient course as the reaction of the synthase progressed, rapidly rising to a maximum of 0.7 microM in the first 10 s, and then declining slowly, reaching 0.1 microM after 60 s. The maximal level of PGG2 achieved during the reaction was constant at about 0.7 microM with higher amounts of added cyclooxygenase capacity (0.3-0.6 microM PGG2/s) but was only about 0.4 microM when the added cyclooxygenase capacity was 0.1 microM PGG2/s. The peroxidase was found to lose only 30% of its activity after 90 s, a point where the cyclooxygenase was almost completely inactive. These results support the concept of a burst of catalytic action from the cyclooxygenase and a reactive, more sustained, catalytic action from the peroxidase during the reaction of the synthase with arachidonic acid.