We previously reported the drug interactions of thalidomide with midazolam and cyclosporine A using recombinant human cytochrome P450 3A enzymes and liver microsomes. Heterotropic cooperativity of human P450 3A5 mediated by thalidomide was shown in these in vitro systems. The purpose of this study was to investigate these drug interactions in cultured human liver cell line system more closely resembled livers. A new hepatic cell line system with differentiated HepaRG cryopreserved (Biopredic International, Rennes, France) was adopted. After thawing and culturing of differentiated HepaRG cells cryopreserved, typical oxidation activities were analyzed with midazolam (CYP3A4), omeprazole (CYP2C19), dextromethorphane (CYP2D6), and diclofenac (CYP2C9) by LC with tandem MS spectrometry system. These marker drug oxidation activities of HepaRG cells tended to be increased up to 1 week gradually and maintained for 2 weeks. Under these conditions, midazolam 1'-hydroxylation activity was suppressed by 50 % in the presence of 100 µM thalidomide while no effects were seen on midazolam 4-hydroxylation. On the contrary, both midazolam 1'- and 4-hydroxylation activities were suppressed by 100 µM of 5-hydroxythalidomide, a primary human metabolite of thalidomide. These results suggest that cultured human cell line system with differentiated HepaRG cryopreserved could be applicable to thalidomide drug interaction study. Further oxidative metabolism of 5-hydoroxythalidomide to 5-hydroxythalidomide GSH-conjugate formation is under investigation using the new HepaRG cell system mimicking in vivo human livers.