Lactoferrin has repeatedly been proposed to be a target for antineutrophil perinuclear cytoplasmic antibodies (P-ANCA), which are present in 67% of ulcerative colitis (UC) cases. However, this high prevalence has not been achieved with either Western blots or monospecific ELISA on the basis of purified lactoferrin bound to the solid phase. We reevaluated autoantibodies against lactoferrin by indirect immunofluorescence (IIF), using a lactoferrin-tuned granulocyte substrate. Slides with ethanol-fixed human granulocytes were stripped of their P-ANCA targets by high-salt treatment and then reconstituted with human lactoferrin (LFR granulocytes). The slides were then subjected to IIF with a panel of sera (39 UC, 10 antimyeloperoxidase-positive vasculitis, 50 healthy blood donors). The human sera were also analyzed with antilactoferrin ELISA. In 28 of 39 (71.8%) sera from UC patients, antibodies could be determined that bound exclusively to LFR granulocytes. Nuclease-treated cells failed to show this reactivity. ELISA detected antilactoferrin antibodies in only two UC sera. Lactoferrin is a major P-ANCA target in UC but requires DNA to present the epitopes relevant for the reaction with the autoantibodies. Antigen-stripped and lactoferrin-reconstituted granulocytes can be used in IIF to diagnose antilactoferrin antibodies in UC more reliably than with existing ELISA systems.