An increase in intracellular Ca2+ occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca2+ extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca2+. To test this possibility, Ca2+ pump phosphorylation by [γ-32P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30s with Ca2+, the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca2+ plus La3+, in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50–90s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca2+-dependent radioactive band of about 150kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca2+ pump units during cell ageing, thus indicating that the reduced Ca2+ pumping activity of aged cells does not arise from pump proteolysis.