Abstract Background Murine hindlimb reperfusion injury (I/R), is initiated by activation of the classical pathway of complement. Complement receptor-2 knockout mice (Cr2 -/-) are protected from I/R injury due to defective B-1 cells with a resulting deficient natural immunoglobulin M (IgM) repertoire. Cr2 -/- and wild type (WT) mice were studied to isolate the antibody or antibodies responsible for initiation of I/R. Methods IgM-secreting B-1 cell clones were produced with hybridoma technology from WT cells. Of 21 clones tested in murine I/R models, only 1 clone, CM22, was found to restore injury in protected mice. Cr2 -/- mice reconstituted with IgM from individual clones, WT serum, or saline were subjected to 2 hours hindlimb ischemia and 3 hours reperfusion and compared with WT. Results Muscle injury in Cr2 -/- mice reconstituted with CM22 was similar to injury in WT mice reconstituted with saline and Cr2 -/- mice reconstituted with WT serum. This injury was 137% greater ( P < .05) than in both Cr2 -/- mice reconstituted with saline and those reconstituted with a different IgM clone, CM31. IgM and C3 deposition was found only on injured muscle of WT mice or Cr2 -/- mice reconstituted with CM22 or WT serum. Conclusion A single clone of self-reactive IgM, CM22, can initiate complement-dependent I/R injury.