Our previous study identified a vincristine‐selected multidrug resistance (MDR) cell line, HOB1/VCR, derived from a lymphoblastoma HOB1. The HOB1/VCR cells are resistant to typical MDR drugs and are cross‐resistant to P‐glycoprotein‐independent drugs such as cisplatin (cis‐diamminedichloroplatinum [II]). The mechanism of this atypical MDR phenotype is uncertain. The present study provides evidence regarding the contribution of reactive oxygen species (ROS) to the resistance of cells in response to treatments (vincristine, cisplatin and H₂O₂). Notably, the HOB1/VCR cells were cross‐resistant to H₂O₂. High levels of ROS formed in both sensitive and HOB1/VCR cells by H₂O₂, and moderate levels of ROS were generated by treatment with cisplatin and vincristine. The ROS level in HOB1/VCR cells was lower than that in sensitive cells following treatments. The ROS level was reduced markedly by a non‐toxic concentration of N‐acetyl‐l‐cysteine, a ROS scavenger, in drug‐treated cells, and was correlated with reduced cytotoxicity. Furthermore, concentrations of glutathione and glutathione peroxidase, but not superoxide dismutase and catalase, increased in HOB/VCR cells. The dl‐buthionine‐[S,R]‐sulfoximine inhibited formation of glutathione and sensitized both cell types to treatments. Therefore, overexpression of an H₂O₂‐reducing system, glutathione–glutathione peroxidase, has a role in resistance. Experimental results further demonstrate that ROS is likely a primary signal in the acquisition of the MDR phenotype and therefore a potential target when designing drugs for chemoresistance. (Cancer Sci 2007; 98: 1206–1214)