Microheterogeneity of antithrombin III (AT-III) was investigated by crossed immunoelectrofocusing (CIEF) on eleven molecular variants. A normal pattern was found in five variants while two different abnormal CIEF patterns were found in the other four and two variants, respectively. Point mutations causing a major pIchange (exceeding 4.0) of the amino acid substituted lead to alterations in the overall microheterogeneity. The variants thus substituted share a first type of abnormal CIEF pattern with alterations throughout the pH range, regardless of the location of the mutation (reactive site and adjacent regions or heparin binding region). Minor amino acid pI changes in these regions do not alter the AT-III overall microheterogeneity, whatever the resulting functional defect. However, if the mutation is placed in the region around positions 404 or 429, then even minor changes of the amino acid pI seem able to alter the overall charge, leading to a second type of abnormal CIEF pattern with the main alteration at pH 4.8–4.6. Neuraminidase treatment leads to disappearance of microheterogeneity except for the variants with the Arg393 to Cys substitution. Addition of thrombin induces CIEF modifications specifically related to the functional defect. A normal formation of thrombin–antithrombin complexes induces a shift towards the more acid pH range, whereas in the variants substituted at the reactive site the CIEF pattern is substantially unaffected by thrombin; variants substituted at positions 382–384 show a maximal thrombin-induced increase of the isoforms at pI 4.8–4.6. Therefore mutant antithrombins with different functional abnormalities but sharing a common CIEF pattern were well distinguished. Thus CIEF can be a useful tool to investigate congenital AT-III pathological variants as well as different functional roles of physiological isoantithrombins.