Treatment of NIH3T3 cells with the tumor promoter phorbol-12-myristate-13-acetate (PMA) results within 30 min in a 1.8-fold elevation of xanthine oxidase (XO) activity, an enzyme capable of generating reactive oxygen species such as superoxide and hydrogen peroxide. Simultaneous administration of 2 and 10 microM curcumin with 100 ng/ml PMA inhibits PMA-induced increases in XO activity measured 30 min later by 22.7% and 36.5%, respectively. The PMA-induced conversion of xanthine dehydrogenase (XD) to XO is reduced by curcumin to the basal level noted in untreated cells. Activity of XO is remarkably inhibited by curcumin in vitro, but not by its structurally related compounds caffeic acid, chlorogenic acid and ferulic acid. Based on these findings, induction of XO activity is deemed to be one of the major causative elements in PMA-mediated tumor promotion, and the major inhibitory mechanism of curcumin on PMA-induced increases in XD/XO enzyme activities is through direct inactivation at the protein level.