During the course of the study on the oxidative damage in fishes, we previously revealed that the primary target organ of ozone toxicity to fish was not gill to cause gill injury but rather red blood cells (RBC). However, the mechanism of ozone toxicity to RBC at the molecular level has remained unrevealed. In the present study, we examined whether hemoglobin (Hb) or hemoglobin-derived iron (HbFe) could participate in oxidative damage of rainbow trout (Oncorhynchus mykiss) RBC induced by ozone exposure. Carbon monoxide (CO) was used to convert the Hb to a stable derivative prior to ozone exposure and the effect of free ferrous irons from Hb as a result of ozone oxposure was examined by the addition of diethylene triamine pentaacetic acid (DTPA) in the medium. Ozone oxposure induced hemolysis, membrane lipid peroxidation, decrease of antioxidative substances and activity of acetylchoinesterase (AChE). Those damages were not suppressed by the addition of DTP A but were effectively supressed by incubating RBC with CO. Ozone or ozone derived species should penetrate through the membrane without direct membrane damage and react with the iron of Hb to genarate hydroxyl radical or other reactive species inside RBC. No inhibition of DTP A addition suggests that ozone exposure did not release iron from Hb. These results indicate that Hb itself performs an important role in the oxidative damage of RBC membrane; i.e., oxidative damage was caused by the reactive oxygen species generated inside RBC rather than by direct attack of extracellular direct ozone attack to RBC outer membrane.