Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120-day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor MetHb formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that MetHb binding to the cytoplasmic domain of band 3 in peroxidized, but not in untreated erythrocyte membranes, induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density-gradient procedure, have enabled us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ~ 0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced NADH-dependent reductase activity, and accumulated oxidative membrane damage. These findings have enabled us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably due to its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from the band 3, enabling the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.