Fully differentiated 3T3-L1 adipocytes, maintained in the presence of insulin, exhibit up-regulation of insulin-binding capacity when insulin is removed from the culture medium. Both cell surface and total cellular insulin receptors increase by 1.8- to 2.0-fold during the 24-h period following the removal of insulin. When up-regulated 3T3-L1 cells are exposed to 10(-8) M insulin down-regulation of insulin receptors occurs with a t1/2 of 2-3 h. Down-regulation was complete after a 10-h exposure to insulin and resulted in a 50-60% decrease in levels of cell surface and total cellular insulin-binding capacities, respectively. Scatchard analysis revealed that these changes in insulin binding are due to an alteration of receptor number and not insulin-binding affinity. To clarify the mechanism(s) by which the regulation of insulin receptor level occurs, rates of receptor synthesis and degradation were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of up-regulation or down-regulation. Up-regulation, however, caused an increase in receptor half-life from 8.1 h in the control cells to 14.8 h. Subsequent down-regulation brought about a return of receptor half-life to 6.9 h. These results indicate that insulin-dependent regulation of insulin receptor level in 3T3-L1 adipocytes involves a change in the rate of receptor degradation. Further studies indicated that regulation of insulin receptor level has physiological significance, since up-regulated cells exhibit an increased responsiveness of 2-deoxyglucose uptake to insulin compared to down-regulated cells.