SummaryConsiderable attention has been focused recently on α2-macro-globulin (α2M), a major endopeptidase inhibitor in blood plasma, as a possible source of the primary defect in cystic fibrosis (CF). We report here studies designed to compare the structure of CF α2M with normal α2M to determine if there is a difference. The physicochemical properties of purified α2M as revealed by various electrophoretic techniques, covalent proteinase binding properties, and primary structural studies on a variety of partial hydrolyzates of CF α2M and normal α2M are compared. These studies were carried out on eight different individual isolates of CF α2M and three age-matched normal α2M preparations and α2M isolated from fetal cord blood. Three properties of CF α2M were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): (1) the existence of four identically-sized subunits in the native molecule (10), (2) the cleavage of this subunit into fragments of approximately 100,000 daltons upon interaction with proteinases (10), and (3) the cleavage of an alkaline/heat sensitive bond to produce 120,000 and 60,000 dalton fragments (11). Both CF and normal α2M were cleaved to the extent of 79–87%. CF α2M behaves identically with normal α2M with regard to all these properties. Salvesen and Barrett (24) have demonstrated that varying proportions of several [125I]-labeled proteinases form SDS-stable, non-reducible links to normal α2M. Two of the CF α2M preparations were studied to determine if similar covalent binding of proteinases occurred. The positions of the labeled and % of proteinase bound bands in SDS/reduced PAGE system were identical for normal α2M and CF α2M. These results indicate that CF α2M behaves normally with regard to covalent binding of proteinases. Qualitative comparison of the peptide fragments separated by SDS-PAGE or isoelectric focusing of CF and normal α2M produced by partial proteolysis with trypsin, chymotrypsin orStaphylococcus aureusV-8 proteinase did not reveal any differences unique to CF α2M. The cyanogen bromide fragmentation studies and the cysteine cleavage studies also indicated that no major change in the positions of methionyl residues or cysteinyl/cystinyl residues has occurred in CF α2M. The failure of all these different studies and those reported by others to demonstrate any differences between CF and normal α2M makes it highly unlikely that there is a primary defect in α2M in CF.