The γ‐carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser‐induced fluorescence (CE‐LIF). Both plasma‐derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium‐dependent conformational change that is required for prothombinase function. Thus, the CE‐LIF assay is useful in determining the carboxylation status of recombinant proteins.