Summary: Insulin-binding antibodies in human sera were studied with respect to their immunoelectrophoretic and chromatographic properties. The reagents used for developing precipitate arcs in immunoelectrophoresis were mixtures of a trace amount of I131 insulin and rabbit antiserum against particular components of human serum. Arcs formed by reaction of rabbit antibody and insulin-binding components of human serum fix I131 insulin and can be revealed by radioautography. The method is simple and extremely sensitive. In addition, the nature of insulinbinding components can be readily identified by comparing the radioautograph with the stained slide. Fifteen out of 18 sera from diabetic patients under insulin treatment contained detectable amounts of insulin-binding components, presumably antibodies. (Three negative sera had been obtained from patients receiving less than 30 units insulin daily.) Sera from three insulin-allergic patients also contained insulin-binding antibodies. None of the sera from normal donors and diabetic patients who had never received insulin showed such components. In the majority of the sera tested, the insulin-binding activity was found only in the γ-globulin. However, a serum from an insulin-allergic patient had the activity in the β2A- and β2M-globulins in addition to the γ-globulin. Another serum from an insulin-insensitive patient (requiring 90 units daily) also showed the detectable activity in the β2A-globulin. Antibodies of the γ-globulin type were further separated into two distinctly different fractions by chromatography on diethylaminoethyl cellulose. These two fractions differed in their electrophoretic mobility (migrating in the γ1- and γ2-globulin regions respectively), but reacted in an identical manner against rabbit antiserum. The binding characteristics of these fractions to I131 labeled bovine zinc insulin were quite similar to each other.