We previously demonstrated that brain cyclooxygenase (COX)-2 mRNA and protein levels, and prostaglandin E2 (PGE2) level, are down-regulated in cytosolic phospholipase A2 (cPLA2) -deficient mice. To further investigate the interaction between upstream and downstream enzymes involved in brain prostaglandin synthesis, we examined expression and activity of COX-1, of different PLA2 enzymes and of prostaglandin E synthase (PGES) enzymes in COX-2(-/-) mice. We found that the PGE2 level was decreased by 51.5% in the COX-2(-/-) mice brains, indicating a significant role of COX-2 in brain formation of PGE2. However, when we supplied exogenous arachidonic acid (AA) to brain homogenates, COX activity was increased in the COX-2(-/-) mice, suggesting a compensatory activation of COX-1 and an intracellular compartmentalization of the COX isozymes. Consistent with COX-1 increased activity, brain expression of COX-1 protein and mRNA also was increased. Activity and expression of cPLA2 and secretory PLA2 (sPLA2) enzymes, supplying AA to COX, were significantly increased. Also, the PGE2 biosynthetic pathway downstream from COX-2 was affected in the COX-2(-/-) mice, as decreased expression of microsomal prostaglandin E synthase-2 (mPGES-2), but not mPGES-1 or cytosolic PGES, was observed. Overall, the data suggest that compensatory mechanisms exist in COX-2(-/-) mice and that mPGES-2 is functionally coupled with COX-2.