The non-steroidal antiandrogen flutamide is widely used for treatment of prostatic cancer, but causes side effects, including cholestatic hepatitis and fulminant hepatitis. We investigated the pathogenesis of flutamide-induced cholestatic hepatitis, focusing on the bile salt export pump (BSEP; ABCB11), which exports bile salts to the bile. We examined the inhibitory effects of flutamide and its active metabolite, hydroxyflutamide, on the transport of taurocholic acid (TCA) by membrane vesicles derived from hBSEP-expressing Sf9 cells. Flutamide inhibited the transport of TCA by hBSEP (IC50 value, about 50 microM), while hydroxyflutamide had no effect at up to 100 microM. When flutamide was administered to rats as a single oral dose of 100 mg/kg, the biliary excretion rate of bolus-injected [3H]TCA was decreased and the liver tissue concentration of flutamide exceeded 50 microM. Repeated doses of flutamide for 5 d (10 mg/kg/d) also decreased the biliary excretion rate of bolus-injected [3H]TCA. In this case, the liver tissue concentration of flutamide was below 0.1 microM. In both cases, no change in the mRNA level of rat Bsep was detected by RT-PCR. These results suggest that flutamide itself, but not its major metabolite, may cause cholestasis by inhibiting BSEP-mediated bile salt excretion.