In isolated tubular segments (ITS) of rat kidney cortex, we studied the effect of hemoglobin (Hb) on reoxygenation damage. All tubules were suspended in Ringer's solution containing 5-mm glycine and oxygenated for 30 min with 95% O(2):5% CO(2), followed by a 30-min period with 95% N(2):5% CO(2), and final reoxygenation for 60 min. Untreated tubules served as controls. Different concentrations of free Hb and equivalent amounts of intact erythrocytes were added to the incubation medium. Secondly, we added deferoxamine (DFO) to Hb and erythrocytes. Membrane leakage and lipid peroxidation were measured by lactate dehydrogenase and glutamate dehydrogenase and the development of thiobarbituric acid reactive substances. Cell function was quantified by gluconeogenesis and intracellular potassium accumulation. Hb exerted concentration-dependent cytotoxic effects indicated by significantly increased enzyme leakage rates, lipid peroxidation and a significantly decreased cell function (P < 0.05), in ITS during hypoxia, and subsequent reoxygenation. Moreover, we found that toxicity of both Fe(2+) and Fe(3+) ions increased with rising concentration. However, Fe(2+) showed a higher tissue toxicity than Fe(3+). DFO reduced significantly the reoxygenation damage of free Hb and iron ions. Our data clearly demonstrate a pronounced cytotoxic effect of free Hb in ITS, which critically depended on the reduction state of the iron ions.