The purpose of this study was to investigate the role of superoxide anion(O2-) in the regulation of epidermal growth factor (EGF) or epidermal growth factor receptor (EGFR) expression and proliferation in the prostate cancer cell line PC3. Cell proliferation was tested by a 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay in the presence of O2-, EGF or their combination. Immunohistochemistry was carried out to assay the expression of EGF or EGFR. EGF or EGFR mRNA expression in the cells treated with O2- was examined by in situ hybridisation. The proliferation was significantly inhibited by O2- in a concentration-dependent manner ranging from 9 to 36 micromol/l nicotinamide adenine dinucleotide (NADH) combined with 2-8 micromol/l N-methylphenazonium methyl sulfate (PMS). The enhancement of proliferation induced by 5 ng/ml EGF was significantly overcome by O2-. Although O2- was not able to alter EGFR mRNA expression, O2- at the concentration of 18 micromol/l NADH and 4 micromol/l PMS reduced EGFR protein expression. O2- at the concentration of 18 micromol/l NADH and 4 micromol/l PMS can downregulate EGF and EGF mRNA expression.