It has been shown that the conjugate of the fragment A of diphtheria toxin to insulin is cytotoxic to cultured cells bearing insulin receptors, apparently through the endocytosis of fragment A. We examined the effect of autoantibodies against insulin receptors on the cytotoxicity of the conjugate. The conjugate was cytotoxic to a rat fibroblast cell line that was resistant to the intact toxin, and the cytotoxicity was inhibited by exogenous insulin, indicating that the fragment A underwent endocytosis through insulin receptors. Immunoglobulins from three patients with type B syndrome of insulin-resistant diabetes blocked the cytotoxicity of the conjugate to Chang's liver cells in a dose-dependent manner. When the cells were pretreated with the immunoglobulins, cytotoxicity of the conjugate was also blocked. These results suggest that autoantibodies against insulin receptors interfere with the binding of the conjugate to insulin receptors or with the endocytosis of fragment A after binding. This assay system seems useful for detecting autoantibodies against the determinants that are involved in the internalization of the ligand-receptor complex.