Antibodies against insulin in three cases of insulin resistant diabetes mellitus were studied. These were IgG antibodies and their insulin-binding capacity was determined by a double antibody precipitation technique. These anti-insulin antibodies were associated in one case with lymphadenopathy, responsive to corticosteroids; anti-insulin activity was detected in an in vitro lymph node culture. In the other two cases, insulin antibodies were associated with an acute viral disease and intermittent insulin resistance of long duration. Although the insulin-binding activity as determined by the double antibody system was reproducible with individual serum samples, serum insulin-binding activity varied markedly on different days. This was presumptively because of variable degrees of free antibody associated with insulin administration; variable amounts of insulin-anti-insulin complexes were present in these sera. Cross-reactivities of insulins and certain insulin fragments from different species were compared by an inhibition technique and they demonstrated the importance of the secondary and tertiary structures in the antigenic determinants of insulin.