To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.