Syngeneic Fischer rat kidneys repeatedly injected with lipid A induced a cytotoxic antibody to cultured syngeneic rat kidney tubular cells. To test for antibodies in the serum of immunized animals, we disaggregated syngeneic rat kidney tubular cells with collagenase and trypsin and established them in short-term culture. Cultured kidney tubule cells were then radiolabeled 24 hours later with chromium 51 and cultured for an additional 24 hours. Rabbit antirat kidney tubule cell antibody served as the positive control antisera in a complement-dependent antibody cytotoxicity assay. Serum samples from animals whose kidneys were innoculated with Re glycolipid were then tested for antibody cytotoxicity. Autoantibody to syngeneic cultured kidney tubule cells was presented in the serum from these animals (P less than 0.01) as well as in the serum of animals injected i.p. with Re glycolipid when compared with saline controls. The cytotoxic antibody could be removed by absorbing with syngeneic cultured kidney tubule cell membranes. These results suggest that the glycolipid from the mutant strain Re 595 of Salmonella minnesota stimulates a crossreactive antibody to cultured rat kidney tubular cells. The methodology used in these experiments provides an in vitro models for investigating the importance of the immune system in the pathogenesis of pyelonephritis.