We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.