The glycolethers 2-methoxyethanol (2-ME), 2-ethoxyethanol (2-EE), and 2-butoxyethanol (2-BE) are used as solvents and have teratogenic, spermatotoxic, and hematotoxic effects. These glycolethers are oxidized to their corresponding alkoxyacetic acids, probably by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). This metabolic conversion of the glycolethers is a prerequisite for development of toxicity, as the toxic effects have been shown to be due to the alkoxyacetic acid metabolites. Three isoenzymes of ADH have been detected in rat tissues. The liver contains two of these isoenzymes, ADH-2 and ADH-3. It has also been shown that the activity level of ADH is strongly sex dependent, with higher activity in females than in males. In the present study, we have investigated whether one or both of the ADH isoenzymes in male and female rat livers were able to oxidize 2-ME, 2-EE, and 2-BE and whether one or both of the ADH isoenzymes in male rat liver were able to oxidize 2-pentyloxyethanol and 2-hexyloxyethanol. Our results indicated that only the ADH-3 isoenzyme effectively oxidized the glycolethers in rat liver. Both ADH-2 and ADH-3 were able to oxidize medium chain aliphatic alcohols with a chain length corresponding to the glycolethers. The activity of ADH is higher in female than in male rat liver. However, it was the same ADH isoenzyme (ADH-3) that oxidized the different glycolethers tested in both male and female rat livers, and the substrate specificity was 2-BE > 2-EE > 2-ME.