Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G(2) (PGG(2)) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG(2) to PGH(2). Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with greater than or equal to60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.