Insulin binding and down-regulation were studied in primary cultures of human and rat hepatocytes. Equilibrium binding characteristics were similar in the two species, with a curvilinear Scatchard plot compatible with binding sites of high and low apparent affinities. The dose-response curve for insulin stimulation of glycogen synthesis coincided with the dose-occupancy curve of the low affinity sites; a maximal biological effect was reached at 50% occupancy. Exposure of rat hepatocytes to 2 × 10⁻⁹M insulin for 24 h produced a 48% decrease in binding capacity due to decreases in both types of binding sites and a 50% decrease in maximal insulin stimulation of glycogen synthesis. After exposure to the same insulin concentration human cells had an 83% decrease in maximum binding capacity, due exclusively to a complete loss of low affinity sites, and a total suppression of insulin stimulation of glycogen synthesis. In both species there was a biphasic relation between degradation and binding: over the range of insulin concentration producing binding mainly to high affinity sites degradation increased slowly as binding increased; with higher insulin concentrations and saturation of high affinity sites degradation increased rapidly as binding to low affinity sites increased. At equal levels of binding, down-regulated cells degraded insulin more rapidly than normal cells. It is concluded that: 1) insulin bound to sites of low apparent affinity is responsible for the hormone’s glycogenic effect, 2) down-regulation of human hepatocytes virtually eliminates such binding and the glycogenic response and also increases the rate of degradation of insulin in relation to the amount bound to high affinity sites, 3) human cells are more sensitive than rat cells to down-regulation. It is suggested that in human cells the major effects of exposure to insulin are an inhibition of insulin internalization and an increase in the rate of degradation of that insulin which is internalized; in rat cells the major effects are a decrease in cell surface binding and an increased rate of degradation of internalized insulin. (Endocrinology114, 37, 1984)