Prostaglandins are synthesized by prostaglandin H synthase (PGHS) 1 and 2. PGHS2 is regulated through inducible expression. We report here the regulation of PGHS1 activity by substrate-dependent cooperative activation. The cooperativity is characterized by a Hill coefficient of 1.29 +/- 0.06, a curved Eadie-Scatchard plot, and activation by low concentrations of competitive inhibitors. The activation also appears to induce a conformational change in the cyclooxygenase site. The cooperativity produces a 2-4-fold greater rate of PGHS2-dependent prostaglandin formation compared with PGHS1-dependent prostaglandin formation at arachidonic acid concentrations below 0.5 microM. A consequence of the PGHS1 cooperativity is that the affinity of many cyclooxygenase inhibitors for PGHS1 decreases in parallel to the activation by arachidonic acid. In contrast, the affinity of these inhibitors for PGHS2 is unaffected by the changes in arachidonic acid concentration. This results in a dramatic difference in PGHS2/PGHS1 selectivity at different arachidonic acid concentrations.