SummaryMolecular heterogeneity of antithrombin III (AT III) was investigated by a technique of crossed immunoelectrofocusing (CIEF) in plasma samples of patients from 16 families with AT III congenital defect, including 8 AT III molecular variants. The AT III CIEF pattern was normal in all the patients with AT III quantitative deficiency, showing a balanced decrease of all the peaks. Out of the 8 AT III variants investigated, 6 had an abnormal pattern: the three variants with defective binding to heparin (AT III Roma, AT III Barcelona, AT III Malmö) shared a similar abnormal pattern; three variants with defective binding to serine proteases (AT III Pescara, AT III Milano, AT III Tampere) had a common abnormal pattern clearly different from the first one, whereas the other two variants deficient in the inactivation of the serine proteases (AT III Chicago, AT III Milano 2) showed a normal pattern. The first type of pathological pattern (type Roma) was characterized by the presence of an abnormal peak overlapping the normal isoforms present at pH 4.8-4.6 and by an additional peak at pH 4.5. The second type of pattern (type Pescara) showed an additional peak at pH 4.5 and an abnormal quantitative distribution of the isoantithrombins all throughout the pH range (5.2-4.6). In order to separate the abnormal AT III fraction from the normal one, plasma of a patient with Roma defect and serum of a patient with Pescara defect were passed throughout an heparin-ultrogel column. The CIEF of the different AT III Roma and AT III Pescara fractions obtained by affinity chromatography confirmed that the abnormalities found in the corresponding patterns of the native samples were related to the pathological isoantithrombins only. It was concluded that the CIEF can be an useful tool to characterize abnormal antithrombins and can reveal close affinities among AT III molecular variants belonging to different subgroups according to the conventional tests.