A method was developed for maintaining isolated rat adipocytes in primary culture in a viable state for several days. Using this method, the kinetics of insulin receptor biosynthesis and membrane incorporation were assessed by inactivating (>95%) cell-surface insulin receptors with trypsin and then incubating cells in medium at 37°C for various times up to 44 h. The insertion of nascent receptors, into the plasma membrane was monitored by measuring the recovery of specific 125l-insulin binding activity. After trypsinization, insulin binding activity recovered at a linear rate with half of the original cell-surface receptor complement replenished by 24 h (6000 receptors or 2% of total surface receptors/h). This recovery was most probably mediated by de novo receptor synthesis, since no recovery of specific insulin binding was seen when cells were pretreated with cycloheximide for 3 h. Under conditions where protein synthesis was blocked immediately after trypsinization, however, a lag of 2–3 h was observed before receptor incorporation was inhibited. This lag suggests that insulin receptors are processed and routed within the cell for 2–3 h before being inserted into the plasma membrane. In support of this hypothesis it was found that the intracellular pool of insulin receptors (10% of the total receptor complement) could be depleted by about 80% by treating cells with cycloheximide for 3 h. Thus, it appears that most, if not the entire, intracellular receptor pool in adipocytes consists of newly synthesized receptors en route to the cell surface. Insulin, after binding to cell-surface receptors, triggers various biologic actions in adipocytes such as glucose transport and endocytosis of insulin-receptor complexes. To determine whether nascent receptors are functional immediately on insertion, we compared the recovery of insulin binding activity after trypsinization with the ability of these cells to respond to insulin with an increase in glucose uptake and an augmented rate of insulin internalization. These data revealed a high correlation between recovery of binding and biologic responsiveness. Thus, these data indicate that immediately upon insertion, or shortly thereafter, receptors become functional and are able to mediate the biologic actions of insulin.