A simplified approach for estimating free and total anti-insulin antibodies and soluble insulin-anti-insulin immune complexes in serum has been developed and evaluated. For determination of free anti-insulin antibodies, a binding ratio for 125I-labeled insulin at fivefold serum dilution is calculated; the binding ratios obtained are reproducible (run-to-run coefficient of variation, 7.3%) and correlate well with titration of anti-insulin antibodies by other techniques (correlation coefficient, 0.9327). Total anti-insulin antibody concentrations are determined by calculating the [125I]insulin binding ratio for a sample previously acidified, adsorbed with acid dextran-coated charcoal, and neutralized. The difference in binding ratios between two aliquots of the same serum, one diluted 10-fold without manipulation and the second studied at an identical dilution after acidification and removal of free insulin, is taken as an index of the presence of soluble insulin-anti-insulin immune complexes. Because the tests can be performed at a single dilution, both time and materials are conserved without apparent loss of discrimination between sera with high antibody titers and high concentrations of insulin-anti-insulin immune complexes and sera with negative titers or low concentrations.