The nature of hemolytic effect induced by ethylene glycol alkyl ethers was analyzed taking into account G-6-PDH activity, ATP, pyruvate and thiols levels in peripheral blood of rats treated with single doses of 2-ethoxyethanol and 2-butoxyethanol. In addition, the susceptibility to autoxidation of rat erythrocyte lipids was evaluated. A decrease of ATP level in a dose-dependent manner and an increase in protein- and nonprotein-bound sulfhydryl groups were observed. These results indicate that an acute hemolytic effect of ethylene glycol alkyl ethers is not associated with alterations in G-6-PDH activity and the susceptibility of erythrocyte lipids to autoxidation. Increases in protein- and nonprotein-bound sulfhydryl groups seem to indicate the selective hemolysis of the aged erythrocytes. The increase in pyruvate and thiol levels may protect erythrocytes against the appearance of oxidative stress.