An efflux function induced by multidrug resistance-associated proteins (MRPs) contributes to the mechanisms of resistance to methotrexate (MTX). Since it has been reported that the human Caucasian colon adenocarcinoma cell line (Caco-2) shows high MRPs expression, it is assumed that Caco-2 cells are resistant to MTX. Alternatively, the human fibrosarcoma cell line (HT-1080) has been reported to be highly sensitive to MTX. In this study, the difference in the growth-inhibitory activity of MTX in these cell lines was compared as a characteristic of sensitivity. Subsequently, the characterization of MRP (1-8) mRNAs expression profiles before and after MTX exposure in these cell types was examined with the multiplex reverse-transcription-polymerase chain reaction (RT-PCR) method. It was demonstrated that the expressions of MRP2, MRP3, MRP6, and MRP8 mRNAs in the HT-1080 cells were lower than those in the Caco-2 cells and that only MRP5 mRNA expression was induced in the Caco-2 cells after MTX treatment. The MRP mRNAs expression profiles between these two cell lines could be differentiated widely. The characterization of MRPs profiles might be associated with resistance to MTX in cells. Additionally, this multiplex RT-PCR method would be beneficial for further time-dependence investigations of the MRP mRNAs expression profiles.