We have previously reported that epidermal growth factor (EGF) enhanced the invasive and metastatic abilities of regressor ER-1 cells and that ER-1 cells might acquire stable malignant phenotypes after long-term EGF treatment. In this study, we studied the mechanisms of the malignant phenotype acquisition by long-term EGF treatment.<BR>EGF enhanced the chemotactic response of ER-1 cells toward culture supernatant conditioned with rat lung endothelial cells in a dose-dependent fashion and also enhanced invasiveness into the reconstituted basement membrane (Matrigel^TM) in a similar manner. Furthermore, when ER-1 cells were cultured in the presence of EGF for a month (1M EGF ER-1), they acquired stable invasiveness. In EGF binding assay using ¹²⁵I-EGF, the numbers of EGF receptors were similar for ER-1 cells and 1 M EGF ER-1 cells, which expressed both highand low-affinity receptors. RT-PCR analysis demonstrated that mRNA expression of EGF receptors was similar for both ER-1 cells and 1M EGF ER-1 cells. Drug-resistance tests using c-SST-2 cells (parent cells of ER-1) revealed that colonies resistant to 6-thioguanine and ouabain appeared after long-term EGF treatment (one month).<BR>These results suggest that long-term EGF treatment may affect the genomic alteration of ER-1 cells and that these may acquire stable malignant phenotypes