The enzymatic metabolism of [14C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E2 (PGE2) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F2 alpha (PGF2 alpha) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE2-9-ketoreductase activity. Inhibition of the PGH2 leads to PGE2 isomerase by arachidonic acid or its metabolites could not explain the abrupt half in PGE2 biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE2 biosynthesis while PGF2 alpha production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE2 production at concentrations that did not affect PGF2 alpha biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH2 leads to PGE2 isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH2 leads to PGF2 alpha reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.