Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass = 21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16 +/- 1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of mu- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.