To study changes in glutathione redox status as an indicator of oxidant stress during hypoxia and reoxygenation, we perfused isolated rat lungs with a high or low oxygen perfusate and measured the release of total glutathione and glutathione disulfide (GSSG) into the perfusate. Lungs were perfused for a 20-minute baseline period with a perfusate equilibrated with 95% O2 and 5% CO2 and ventilated with a 95% O2 and 5% CO2 gas mixture. Only very low amounts of oxygen were measurable in this hypoxic perfusate. The lungs were then perfused from a second reservoir containing perfusate equilibrated with 95% N2 and 5% CO2 and ventilated with a 95% N2 and 5% CO2 gas mixture. After the period of hypoxia, the lungs were reperfused with the 95% O2 and 5% CO2 equilibrated perfusate and ventilated with a 95% O2 and 5% CO2 gas mixture for the remainder of the experiment. Glutathione was measured in the perfusate serially throughout the experiment, and lactic dehydrogenase (LDH) was also measured to assess cell membrane rupture during the infusion. GSSG release remained stable in the baseline and hypoxic period but rose significantly in the reoxygenation period, to concentrations approximately two times basal release. Lung tissue concentrations of GSSC also rose in the reoxygenation period. Decreasing lung glutathione reductase activity by pretreating animals with 1,3-bis-2-chloroethyl)-1-nitrosourea (BCNU) increased GSSG release into the perfusate during reoxygenation. We conclude that GSSG formation and release is increased in the lung during the reoxygenation period after lung hypoxia, suggesting the presence of hydroperoxide and free radical metabolism. These data support the hypothesis that alterations in lung metabolism occur during hypoxia that allow free GSSG formation and release during the reintroduction of oxygen.