Hereditary deficiency of antithrombin-III (AT-III), the major heparin cofactor in human plasma, is a well-established cause of recurrent venous thrombosis. Cross-reactivity of heparin cofactor II (HC II) in assays of AT-III may, in some cases, interfere with the ability to diagnose hereditary deficiency of AT-III. For that reason, we have evaluated the interference by HC II in the new DuPont aca antithrombin assay. Response of the assay to purified AT-III and HC II was compared. Inhibition of the bovine thrombin in the assay was sixfold less per molecule of HC II than of AT-III. This level of selectivity should be adequate to prevent misdiagnosis of patients. Analysis of patient samples showed close correlation (r = 0.91) of values from the automated aca assay with those of a manual assay (Coatest antithrombin, Helena Laboratories).