We have previously reported that long-term epidermal growth factor (EGF) treatment enhanced the malignant phenotype of regressor ER-1 cells and might affect the genomic alteration of ER-1 cells. In this study, we examined whether EGF stimulation might induce reactive oxygen species (ROS) in ER-1 cells and whether ROS could affect genomic instability. ROS of ER-1 cells induced by EGF stimulation for 1 month was observed with a confocal laser-scan microscope after 2', 7'-dichlorofluorescein diacetate (DCFH-DA) staining. ROS of ER-1 cells was significantly augmented by EGF stimulation, and ROS spread over the nuclei after stimulation. To study the effect of ROS on ER-1 cells, we measured the amount of glutathione peroxidase (Gpx), radical scavenger enzyme, and 8-hydroxydeoxyguanosine (8-OHdG), which is a mutation indicator. The amount of Gpx was reduced by EGF stimulation and recovered after sodium selenite treatment. Furthermore, to study modulation of the malignant phenotype of ER-1 cells, each treated ER-1 cell was intrapenitorially transplanted into syngenic SHR-rats. Tumorigenicity of ER-1 cells was significantly enhanced by EGF, and selenite treatment completely inhibited the augmentation of tumorigenicity caused by EGF stimulation. This observation was consistent with the in vitro results. Then, to examine the effects of ROS induced by EGF stimulation on the genomic instability of ER-1 cells, DNA fingerprinting assay was done. In this assay, abnormal bands were observed in four clones (4/19 clones) after EGF stimulation. These results suggest that long-term EGF stimulation can affect the genomic alteration of ER-1 cells and that this phenomenon is possibly caused by EGF inducing ROS.