Insulin was tritiated by semisynthetic replacement of the amino-terminal phenylalanine of the B chain with tritiated phenylalanine. At 15°C, (3H) insulin bound to high affinity receptors on IM-9 cultured human lymphocytes with an affinity constant of about 3 × 109 M−1. The Scatchard plot was curvilinear. At 37C, maximal binding occurred after about 15 min of incubation. Binding fell thereafter due to degradation of insulin by the extracellular fluid. The major degradation product after 120 min coeluted with insulin from Sephadex G-50 and was precipitated by anti-insulin antibody but to a lesser degree than intact insulin. It had little or no biologic activity as assessed by binding to IM-9 lymphocytes. The cell-associated radioactivity was also eluted as a single peak on Sephadex G-50. In contrast to the degradation product, this material retained its ability to bind to insulin receptors. We deduce that this cell-associated material contains the entire A chain, most of the B chain, and is probably native insulin. These data show that insulin bound to IM-9 lymphocytes remains biologically intact.