We studied the usefulness of monitoring antineutrophil cytoplasmic antibodies (ANCA) in chronic graft-versus-host disease (cGVHD), a major complication of allogeneic bone marrow transplantation. Antigen-specific ELISA and indirect immunofluorescence (IIF) were used to search for ANCA in 47 allogeneic bone marrow graft recipients who developed cGVHD and in 43 who did not (controls). Eight patients exhibited ANCA IIF positivity in the cGVHD group, but none in the controls. Specificity was confirmed in antigen-specific assays in only two cGVHD patients, both showing antilactoferrin (anti-LF) activity. One of these patients was followed-up, and the antilactoferrin antibodies were found only at the time of active but limited cGVHD. Among three ANCA IIF-positive patients, two had antinuclear autoantibodies and three antineutrophil alloantibodies secondary to blood transfusion, which may have been responsible for false ANCA IIF positivity. It is concluded that ANCA determination is not useful in patients with cGVHD. Polyclonal activation of B lymphocytes could result in ANCA activity during cGVHD. False-positive ANCA could be due to allo-immunization following blood transfusion. Rare patients may present antilactoferrin antibodies of unknown clinical significance.