Cambridge II (A384S) is a highly prevalent antithrombin variant in the British population (1.14 per 1000) and predisposes carriers to a mild but significant increased risk of thrombosis. To determine if the association of Cambridge II with thrombophilia is due to a perturbation of the antithrombin inhibitory mechanism, we expressed and characterized the variant. Antithrombin Cambridge II was found to be normal in its affinity for heparin, its ability to form sodium dodecyl sulfate-stable complexes with factor Xa and thrombin, and its uncatalyzed stoichiometries and rates of inhibition. However, in the presence of full-length heparin there was a 3- and 7-fold increase in stoichiometry of inhibition of factor Xa and thrombin. The stoichiometries were not affected by pentasaccharides, indicating that the inhibitory mechanism of antithrombin Cambridge II is perturbed only in the presence of a bridging glycosaminoglycan. Thus, the vascular localization of antithrombin Cambridge II would render the carrier slightly thrombophilic. The high occurrence of this mutation and its possible propagation from a few founders suggests an evolutionary advantage, perhaps in decreasing postpartum bleeding.