SUMMARY: Monolayer cultures of minimal deviation hepatoma cells (H4-II-E-C3′) bound and degraded insulin specifically, the apparent Ki value for insulin inhibition of both processes being 1 × 10−8 M, indicating that cellbound 125l-insulin is the substrate for subsequent hormone degradation in these cells as in isolated hepatocytes.1 The time course of insulin binding to its receptor depended on hormone concentration and temperature. Degradation of insulin also depended highly on temperature, with little or no degradation occurring at less than 20°C, a temperature below which a membrane–lipid phase transition may block hormone translocation or uptake. The effects of various agents on the binding and degradation of 125l-insulin also were tested. Agents believed to inhibit intralysosomal degradation of various proteins also inhibited the degradation of 125l-insulin by H4 cells (chloroquine, ammonium chloride, procaine, and lidocaine); inhibitors of energy production (dinitrophenol, sodium cyanide) inhibited degradation; an agent which inhibits microtubule function (vinblastine) blocked insulin degradation; and methylamine, reported to prevent receptor aggregation,2 also interfered with insulin processing. These findings are consistent with a model for cellular insulin processing, comprising receptor binding, clustering of receptors, endocytotic uptake, intralysosomal degradation, and extracellular release of some degradation products. H4 cells were highly sensitive to insulin. The KE for a half-maximal response of hormone-stimulated incorporation of 14Cglucose into glycogen was 10−11 M insulin, corresponding to less than 1% receptor occupancy. This response was also mimicked by concanavalin A at a concentration of 10 μug/ml. Vinblastine and chloroquine both significantly inhibited insulin-stimulated glucose incorporation into glycogen without affecting basal levels. However, since these inhibitory effects were not relieved by addition of excess insulin, it seemsunlikely that their action on glycogen synthesis was exerted only at the level of the generation of an active intermediate or degradation product from hormone-receptor complexes. The hormone-sensitive H4 cells thus provide a useful system for further studies examining the role of insulin-receptor uptake in hormone action, receptor regulation, and signal termination.