A rapid semi-homogeneous cyclooxygenase-2 (COX-2) enzymatic assay using scintillation proximity assay (SPA) technology was developed, and 49 ubiquitous plant secondary metabolites were screened for inhibition of COX-2-catalyzed prostaglandin E(2) (PGE(2)) biosynthesis. Assay conditions were optimized with respect to reaction time, amount of antibody, radiolabeled PGE(2), and SPA beads, and the kinetic parameter, K(m), was estimated. The assay was validated with two natural triterpenoids, ursolic and oleanolic acid, known to inhibit COX-2, as well as with four synthetic COX inhibitors, NS-398, rofecoxib, indomethacin, and aspirin. Plant metabolites of different biosynthetic origin representing several substance classes, including alkaloids, anthraquinones, flavonoids, phenylpropanes, steroids, and terpenes, were screened for inhibition of COX-2-catalyzed PGE(2) production. Of these 49 plant metabolites, eugenol, pyrogallol, and cinnamaldehyde (with IC(50) values of 129, 144, and 245 microM, respectively) were found to inhibit COX-2. This study showed that a COX-2-catalyzed PGE(2) assay using SPA is suitable for screening natural compounds with respect to COX-2 inhibition.