Serum samples collected pre- and post-transplant nephrectomy were screened for alloreactive lymphocytotoxins. Three patients demonstrated circulating anti-B cell antibodies with markedly increased cytotoxicity titers after nephrectomy, suggesting that there was active binding of antibodies within the renal allograft. Two of the postnephrectomy serum samples after absorptions with platelets and B cells reacted by immunofluorescence with cultured primary kidney cells but not against B or T cells from the same donor. To clarify further the role of such alloreactive antibodies, eluates were prepared from 14 rejected renal allografts and were analyzed for reactivity to cultured primary kidney cells against different leukocyte targets and endothelial cells. Reactivity toward B cells and kidney cells was demonstrated with 80% of the eluates. Absorption experiments of the positive eluates demonstrated multiple types of alloreactive antibodies. In addition to anti-DR activity, eluates had antibodies reactive to kidney cell antigens, monocytes, macrophages, endothelial cells, and polymorphonuclear leukocytes. The demonstration of multispecific alloantibodies in eluates of rejected kidneys is consistent with an important role(s) in maintenance and/or rejection of the kidney allograft.