1. Hyperbaric oxygen (HBO) is a widely used treatment modality in many diseases. A known side-effect of HBO is the production of reactive oxygen species (ROS). Many anti-oxidants, such as vitamins C and E, riboflavin and selenium, have been used successfully to scavenge the ROS produced by HBO administration. 2. The aim of the present study was to determine whether melatonin, a newly discovered anti-oxidant, has a protective effect against the overproduction of ROS produced by HBO in rat brain tissue. 3. Seventy male Sprague-Dawley rats were divided into seven groups as follows: 1, daytime control; 2, daytime HBO; 3, melatonin; 4, daytime HBO plus melatonin; 5, night-time control; 6, night-time HBO; and 7, night-time HBO under light exposure. 4. Hyperbaric oxygen was administered at 303 kPa for 120 min. Melatonin was injected at a dose of 10 mg/kg, i.p. Brain malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were measured to elucidate oxidant status. 4. The MDA and SOD levels of groups 2 and 7 increased significantly. Exogenous (group 4) and endogenous (group 6) melatonin protected against HBO-induced lipid peroxidation. Exogenously administered melatonin (groups 3 and 4) had increased levels of the anti-oxidant enzymes SOD and GPx. 5. In conclusion, HBO caused oxidative stress and melatonin exhibited protective effects. Both endogenously produced and exogenously administered melatonin were found to be effective.