Raloxifene (Evista) is a second generation selective estrogen receptor modulator used in the treatment of osteoporosis and for chemoprevention of breast cancer. It is bioactivated to reactive intermediates, which covalently bind to proteins and form GSH conjugates upon incubation with NADPH and GSH-supplemented human and rat liver microsomes. Despite these in vitro findings, no major raloxifene-related toxic events have been reported upon its oral administration to humans. This disconnect between safety of raloxifene and its in vitro bioactivation is attributed to its presystemic metabolism via glucuronidation. Current studies investigated the effect of hepatic and intestinal glucuronidation in modulating hepatic availability of raloxifene and its subsequent bioactivation, in vitro. The study design involved preincubation of raloxifene with intestinal microsomes followed by a sequential incubation with liver microsomes. The degree of bioactivation of raloxifene was assessed from the percentage of GSH conjugate formed in liver microsomal incubations or the amount of covalent binding of raloxifene-related material to liver microsomal proteins. The results indicated that human intestinal glucuronidation limited the hepatic exposure of raloxifene that underwent bioactivation in the liver. Similar experiments with rat microsomal preparations showed very little effect of intestinal glucuronidation. This effect of intestinal glucuronidation and the observed species difference were explained by comparing the efficiency (Cl(int)) of glucuronidation and oxidation in the two species. These findings suggested that even though the rate of bioactivation in the two species was similar, the Cl(int) of glucuronidation was 7.5-fold higher in the human intestine as compared to rats. These results support the hypothesis that intestinal glucuronidation modulates the amount of raloxifene undergoing bioactivation by liver and corroborate the importance of assessing other competitive metabolic pathways and species differences in metabolism prior to extrapolation of bioactivation results from rats to humans.