Metabolism of the teratogen thalidomide is proposed to be relevant to its toxicological action. We demonstrated the formation of the glutathione (GSH) conjugate of (R)-5-hydroxythalidomide in vivo in chimeric NOD-scid IL2Rg(null) mice with humanized livers (uPA-NOG mice). After an oral administration of racemic thalidomide (270 mg/kg), plasma concentrations of 5-hydroxythalidomide were significantly higher in humanized mice than in control mice. The GSH conjugate of 5-hydroxythalidomide was detected in the plasma. These results indicate that livers of humanized mice mediate thalidomide 5-hydroxylation and further oxidation leading to the GSH conjugate in vivo as well as in vitro and suggest that thalidomide activation occurs.