Animals that inhabit environments that fluctuate in oxygen must not only contend with disruptions to aerobic metabolism, but also the potential effects of reactive oxygen species (ROS). The goal of this study was to compare aspects of ROS metabolism in response to O2 variability (6 h hypoxia or hyperoxia, with subsequent normoxic recovery) in two species of intertidal sculpin fishes (Cottidae, Actinopterygii) that can experience O2 fluctuations in their natural environment and differ in whole-animal hypoxia tolerance. To assess ROS metabolism, we measured the ratio of glutathione to glutathione disulfide as an indicator of tissue redox environment, MitoP/MitoB ratio to assess in vivo mitochondrial ROS generation, thiobarbituric acid reactive substances (TBARS) for lipid peroxidation, and total oxidative scavenging capacity (TOSC) in the liver, brain and gill. In the brain, the more hypoxia-tolerant Oligocottusmaculosus showed large increases in TBARS levels following hypoxia and hyperoxia exposure that were generally not associated with large changes in mitochondrial H2O2 In contrast, the less-tolerant Scorpaenichthysmarmoratus showed no significant changes in TBARS or mitochondrial H2O2 in the brain. More moderate increases were observed in the liver and gill of O. maculosus exposed to hypoxia and hyperoxia with normoxic recovery, whereas S. marmoratus had a greater response to O2 variability in these tissues compared with the brain. Our results show a species- and tissue-specific relationship between hypoxia tolerance and ROS metabolism.