Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.