Development of resistance to cisplatin in previously treatment-responsive malignancies is a major obstacle to successful treatment. Studies were conducted to investigate acquired resistance to cisplatin in human head and neck cancer cells. The cell lines IMC-3 and IMC-3/CR were studied to assess their respective characteristics of drug accumulation and efflux, cellular glutathione level, and DNA repair. IMC-3/CR cell line was 8.1-fold more resistant to cisplatin than IMC-3 cells. The IMC-3/CR cell line was more efficient at effluxing drugs, which was associated with reduced total drug accumulation and DNA adducts. Expression of MRP and the cellular glutathione level were coordinately increased in the cisplatin-resistant subline. Resistant cells were also 2-fold more efficient at repairing cisplatin-DNA lesions in transfected plasmid DNA. It was concluded from these paired cell lines that drug effluxing by the GS-X pump and DNA repair are the major contributing factors to acquired resistance to cisplatin. Moreover, 2-(4-hydroxyphenyl) ethyl ITC (hITC), which effectively overcomes cisplatin resistance, was discussed.