The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 C. The results demonstrate that insulin is rapidly translocated into the cell's high- and low-density microsomes. Insulin simultaneously induces the translocation of its own receptor into the latter compartment. A protocol has been developed for maintaining isolated rat adipose cells in primary tissue culture. The acute insulin-induced internalization of its own receptor was then confirmed by bioxynthetically labeling cells in culture with either [35S]methionine or [3H]glucosamine. The spatial orientation of the Alpha and Beta subunits of the insulin receptor in the rat adipose cell plasma membrane has been examined using lactoperoxidase/Na125I iodination. The results suggest that the Alpha receptor subunit is primarily exposed on the extracellular surface of the plasma membrane while the Beta receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization. In a preliminary study, anti-insulin receptor antiserum B-10 induces internalization of the insulin receptor and stimulates glucose transport in the rat adipose cell, but does not appear to affect the phosphorylation state of the Beta receptor subunit. Thus, the acute phosphorylation of the insulin is not essential per se for internalization of the receptor or the response to insulin. The biosynthesis of the insulin receptor has been studied in primary cultures of isolated rat adipose cells. The results suggest that insulin receptors are synthesized through the formation and processing of a 190K precursor in the endoplasmic reticulum and Golgi followed by insertion of the mature 135K and 95K subunits into the plasma membrane.