Polyethyleneglycol precipitation of insulin antibodies and insulin-antibody complexes is associated with co-precipitation of part of the free insulin. The recovery of free insulin was found to depend both on the quantity and quality of the antibodies and varied between 94% and 40%. Thus, the concentrations of free and total insulin may be lowered up to 2.5-fold in different sera containing endogenous antibodies. A critical evaluation of these results led to the development of a more reliable method for the assay of total and free insulin, based on individual radioisotopic determinations of the recovery of insulin extracted by PEG. The recovery of insulin added to sera containing antibodies as determined by the present method is within the range of 100 +/1 2.5%. In sera not containing antibodies total insulin concentration was found to be up to 5-times higher than that of IRI and to reach values above 250 muU/ml whereas whereas indirect IRI (i-IRI) may increase over 25 times with age.