Two different isoforms of prostaglandin H synthase, prostaglandin H synthase-1 and prostaglandin H synthase-2, have been identified. Both isozymes catalyze both cyclooxygenase and peroxidase reactions. Residues identified as being essential for catalysis by ovine prostaglandin endoperoxide H synthase-1 are all conserved in prostaglandin H synthase-2. This suggests that the enzymic reaction mechanisms are fundamentally the same for both isozymes. A tyrosyl radical, which may initiate the cyclooxygenase reaction, is detected by electron paramagnetic resonance spectroscopy after addition of arachidonic acid or hydroperoxides to ovine prostaglandin H synthase-1. We report here that human prostaglandin H synthase-2 also generates a tyrosyl radical centered at g = 2.0040 with a width of 29 gauss, similar to prostaglandin H synthase-1. This is the first spectral evidence that the two isoforms are similar mechanistically.