The diagnostic and clinical relevance of Ab to pure and phosphatidylserine-complexed prothrombin for primary and secondary APS was investigated in a total of 357 patients with (n = 169) and without (n = 188) connective tissue diseases. The overall frequency of anti-prothrombin Ab in sAPS, pAPS and patients without APS-related symptoms were found to be 50.0, 37.5 and 22.0%, respectively. From a total of 72 anti-prothrombin-positive samples, 12.5% were specific for pure prothrombin, 31.9% for phosphatidylserine/prothrombin-complexes and 55.6% recognized both antigenic forms. The simultaneous occurrence of other anti-phospholipid Ab was observed in 84% of all sera. Both types of anti-prothrombin Ab are significantly associated with lupus anticoagulant activity, but only Ab to pure prothrombin display such a relationship to clinical manifestations of APS. Based on these results, it cannot be recommended at present to include anti-prothrombin assays in the routine procedure for the serodiagnosis of APS. However, patients negative for lupus anticoagulant and typical APS-related anti-phospholipid Ab should be tested for anti-prothrombin reactivity, favoring, mainly due to its higher specificity, the ELISA containing pure prothrombin as antigen.