In the present study, we investigated the in vitro effect of hypoxanthine on the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase, as well as on thiobarbituric-acid-reactive substances (TBA-RS), in the renal cortex and medulla of rats. Results showed that hypoxanthine, at a concentration of 10.0 μM, enhanced the activities of CAT and SOD in the renal cortex of 15-, 30- and 60-day-old rats, enhanced SOD activity in the renal medulla of 60-day-old rats and enhanced TBA-RS levels in the renal medulla of 30-day-old rats, as compared with controls. Furthermore, we also verified the influence of allopurinol (an inhibitor of xanthine oxidase), as well as of the antioxidants, trolox and ascorbic acid on the effects elicited by hypoxanthine on the parameters tested. Allopurinol and/or administration of antioxidants prevented most alterations caused by hypoxanthine in the oxidative stress parameters evaluated. Data suggest that hypoxanthine alters antioxidant defences and induces lipid peroxidation in the kidney of rats; however, in the presence of allopurinol and antioxidants, some of these alterations in oxidative stress were prevented. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by hypoxanthine. Copyright © 2014 John Wiley & Sons, Ltd.