Apoptotic cells contain nuclear autoantigens that may initiate systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. Heavy chain (H) mutants of 3H9 were constructed, expressed as single chain Fv (scFv) in E. coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine (DOPS) in ELISA and binding was enhanced by B2 glycoprotein I (B2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-B2GPI was achieved by the introduction of arginine residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce may affect the clearance and processing of apoptotic cells.