The identification of panels of tumor antigens that elicit an antibody response may have utility in cancer screening, diagnosis and in establishing prognosis. Until now, autoimmunity in cancer has been mainly revealed in solid tumors. This study was to apply the proteomic approach to the identification of proteins that commonly elicit a humoral response in acute leukemia (AL). Sera from 21 newly diagnosed patients with AL, 20 patients with solid tumors, and 22 noncancer controls were analyzed for antibody-based reactivity against AL proteins resolved by two-dimensional electrophoresis. As a result, autoantibody against a protein identified by mass spectrometry as Rho-GDI2 was detected in sera from 15 of 21 patients with AL (71%). By contrast, such antibody was detected in sera from 1 of 20 patients with solid tumors (5%), and 1 of 22 noncancer controls (4.5%). Other five protein autoantibodies were also found in AL patients with a high frequency and constituted the major target antigens of the AL autoimmune response. The findings of autoantibodies against Rho-GDI2 and others in sera of patients with AL suggest that the proteomic approach we have implemented may have utility for the development of serum-based assay for AL screening and diagnosis.