Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and alpha-2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells. (Blood. 2000;95:1465-1472)