The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A₂ enzymes are sources of cellular arachidonic acid and the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE₂ in the brain. These studies seek to determine the function of cytosolic phospholipase A₂ (cPLA₂) in inflammatory PGE₂ production in the brain. We wondered if cPLA₂ functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1 levels. We investigated these questions in the brains of wildtype and mice deficient in cPLA₂ (cPLA₂^-/-) following systemic administration of lipopolysaccharide (LPS). cPLA₂^-/- mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wildtype mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE₂ concentration of untreated cPLA₂^-/- mice was equal to their wildtype littermates. Following LPS treatment, however, the brain concentration of PGE₂ was significantly less in cPLA₂^-/- than in cPLA₂^+/+ mice (24.4 ± 3.8 ng/g vs. 49.3 ± 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h following LPS. We conclude that cPLA₂ regulates COX-2 levels and modulates inflammatory PGE₂ levels. These results indicate that cPLA₂ inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.