The purpose of the present study was to develop a biochemical marker of inhibition of cyclooxygenase in the central nervous system following oral administration of cyclooxygenase inhibitors. An ex vivo method was developed wherein the brain was removed, incubated at room temperature for 2 min permitting prostaglandins to be synthesized from spontaneously released arachidonic acid. Indomethacin, zomepirac Na, naproxen Na, ibuprofen, aspirin and acetaminophen inhibited the ex vivo production of prostaglandin E2 in a dose-related manner at doses that correlated well with both their potency to inhibit mouse brain cyclooxygenase in vitro, and their antinociceptive potency in a mouse abdominal constriction test. When the brains were frozen immediately after the mice were killed, the above drugs did not reduce the endogenous prostaglandin E2 level. Thus while cyclooxygenase inhibitors did not reduce normal prostaglandin E2 levels in brain, they did attenuate post-mortem increases in prostaglandin E2. This novel assay permits an estimate of cyclooxygenase inhibitory effects in vivo. The data further support the suggestion that many of these drugs may have a central as well as a peripheral effect.