Hemoglobin in stroma-free solution (7 gm/100 ml) was modified by covalently cross-linking the beta-chains with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP). The coupling efficiency was approximately 65%. The oxygen dissociation curve of the coupling mixture was shifted to the right, with a P50 of 30 mm Hg vs. 15 mm Hg for the nonmodified solution. The effect of the modification on tissue oxygenation was studied in the isolated perfused rat liver at normoxia and at hypoxia induced by decrease of flow rate at constant Po2 and hemoglobin concentration. The two perfusates used were a nonmodified hemoglobin solution and the coupling mixture. The chemical modification of the hemoglobin molecule did not affect the vascular resistance in the liver tissue. During normoxia the NFPLP-induced decrease in oxygen affinity was reflected in a higher venous Po2. The differences in the other oxygen-sensitive parameters were not significant. The decrease in O2 supply induced by a decrease of perfusion flow rate (stagnant hypoxia) resulted in a decrease in venous Po2, O2 consumption, and bile flow rate, and an increase in the cytoplasmatic redox level (lactate/pyruvate ratio) and the mitochondrial redox level (beta-hydroxybutyrate/acetoacetate ratio). During hypoxia the changed oxygen affinity of the modified hemoglobin solution was reflected in small but significant differences between both perfusates in the venous Po2 and both redox levels. No change in O2 consumption and bile flow rate was observed. When compared with earlier low-flow perfusions of the isolated rat liver with erythrocytes, the oxygen affinity of hemoglobin solutions appears not to be rate limiting for the O2 consumption, probably because of better tissue perfusion with hemoglobin solutions.