Some immunologic properties of biosynthetic human insulin (BHI) were examined in vitro. An identical behavior was found for BHI and pancreatic human insulin as standard preparation and for A14-mono-labeled BHI and pork insulin as tracer in the insulin radioimmunoassay. BHI proved to be free of human proinsulin and C-peptide. Insulin antibodies in serum of two diabetic patients showed a preferential binding of 125I-bovine insulin. However, the antibody titers were almost identical for A14-mono-labeled BHI and pork insulin. These studies did not reveal any characteristic immunologic properties of BHI compared with highly purified pancreatic human and pork insulin.