Anti-insulin antibodies appear in the sera of patients treated with insulin lastingly. A high anti-insulin antibody level results in the development of insulin resistance. Most of the insulin preparations available on the market contain also glucagon as an impurity. It was therefore to be expected that in part of the patients, who had been treated with insulin lastingly, antibodies would be produced also against glucagon, and the presence of these was actually demonstrated. It is to be assumed that the anti-glucagon antibodies play a role in the pathomechanism of diabetes mellitus, mainly in its labile form. The possible presence of anti-glucagon antibodies must be taken into account when the glucagon concentration in the sera of diabetics is to be determined by means of radioimmunoassay (RIA). The specific antibodies in the serum give false results in the quantitative determination of glucagon. We have tested the sera of 10 diabetics who had been treated with insulin for at least 6 years. All patients were given protamine zinc and crystalline insulin preparations.