Protease inhibitor 10 (PI10) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that is expressed at elevated levels in patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Based upon the ability of the related serpin plasminogen activator inhibitor 2 (PAI-2) to protect cells against tumor necrosis factor alpha (TNFalpha)-induced cell death, this study was initiated to investigate the potential cytoprotective activity of PI10. Two different expression systems (i.e. plasmids encoding either PI10 alone or PI10 fused to the tag: enhanced green fluorescent protein, EGFP) were utilized to stably transfect an eukaryotic model cell system (i.e. HeLa cells) that neither expresses PAI-2 nor PI10. The level of PI10 expression in the stable transfectants was found to correlate with their resistance to TNFalpha-induced cell death. Immunoprecipitation/immunoblotting experiments demonstrated that PI10 is able to form SDS-stable complexes (i.e. M(r) >100,000) with a cytosolic protein(s). Increased levels of the PI10-containing complexes can be detected by TNFalpha treatment by preventing intracellular degradative activities with the proteasome inhibitor N-carbobenzyloxy-leucine-leucine-norvalinal. PI10-containing complexes are dissociated with conditions known to separate classical protease-serpin complexes (i.e., 1.5 m ammonium hydroxide in the presence of SDS). These data support a role for the regulation of intracellular protease activities by ov-serpins.