Male Wistar rats were exposed to ethylene Oxide (Eto) via inhalation at a concentration of 751.7 mg/m3 two hours a day and six days a week for nine consecutive weeks. Sensitivity test of restriction enzymes Mbo I, Sau3A I, Hpa II and Msp I and Southern blot were conducted to study the effects of ethylene oxide on activity of major intracellular modification enzyme, DNA methylase. Results showed that DNA in liver, kidney and testis of rats were completely enzymolysed and only one blot was seen after being treated with Hind III, Mbo I and Sau3A I, and incompletely enzymolysed and multiple blots were seen after being treated with Hpa II and Msp I. There were no significant differences in electrophoresis pattern, number of blots and intensity of radioautograph between the exposed animals and controls. It indicated that no effects of Eto on activity of DNA methylase in the liver, kidney and testis of rats was found, and degree of methylation in the testis was much less than that in the liver and kidney.