Ethylene glycol, best known as antifreeze, is most often ingested accidentally or as a substitute for alcochol by chronic alcohol abusers. The toxicity of ethylene glycol poisoning is due to its toxic metabolites rather than to ethylene glycol itself. In this study, electron spin resonance (ESR) spectroscopy has been used to study free radical generation in rats by acute ethylene glycol poisoning. The radical spin trapping technique was applied where the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) reacted with free radical metabolites to form radical adducts in vivo. The radical adducts from ethylene glycol intoxication were detected in both the bile and urine samples of male Sprague-Dawley rats. The identification of the POBN-(.)[(13)C]ethylene glycol radical adduct provides for the first time direct ESR evidence for the generation of the ethylene glycol-derived radicals during acute intoxication by ethylene glycol, suggesting a new metabolic pathway. Simultaneous administration of alcohol dehydrogenase inhibitor 4-methylpyrazole with ethylene glycol resulted in an enhanced free radical generation in the bile. This report is the first evidence of ethylene glycol free radical metabolism in rats with acute ethylene glycol intoxication.