Summary&agr;2‐Macroglobulin (&agr;2M) is a major plasma protease inhibitor that has been studied because of its suggested role in the pathology of cystic fibrosis (CF). A panel of monoclonal antibodies specific for human &agr;2M were produced and screened for their ability to bind to a number of human &agr;2M samples. We have used these antibodies to characterize individual antigenic sites in this protein. &agr;2M was purified from plasma by polyethylene glycol precipitation followed by zinc chelate chromatography. A total of 23 &agr;2M samples in the native configuration, as well as the nucleophile‐treated configuration, were screened by the panel of 18 monoclonal antibodies in an enzyme‐linked immunosorbent assay procedure. Five of the samples tested were from individuals with cystic fibrosis. &agr;2M from family members of two of these patients was subsequently tested for reactivity with the monoclonal antibodies. One antibody, SAM94, exhibited a significant difference in binding to &agr;2M obtained from CF patients as compared with control individuals. This difference was particularly apparent in the binding of SAM94 to the nucleophile‐treated CF &agr;2M; SAM94 showed significantly reduced binding to four of five unrelated CF individuals (p< 0.005) and three of four cystic fibrosis obligate heterozygotes (p< 0.005).Abbreviations&agr;2M, &agr;2‐macroglobulinCF, cystic fibrosisELISA, enzyme‐linked immunosorbent assayPEG, polyethylene glycolPBS, phosphate‐buffered salinePMSF, phenylmethylsulfonyl fluorideSBTI, soybean trypsin inhibitorTEMED,N,N,N',N'‐tetramethylethylenediamine