This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage.