FT4 assays are based, in part, on assumptions. For example, direct dialysis FT4 assays are assumed to be specific for FT4, based on the assumption that T4 binding proteins (T4BPs) do not cross react in the assay because they are retained during dialysis. The immunoassays that quantify FT4 in dialysates are always non-analog FT4 immunoassays. Today, these assays are based on 125I-T4 binding to, and displacement from, T4 antibody. T4BPs in the dialysates to which they are applied are expected to bind 125I-T4 in the RIA, displacing 125I-T4 from anti-T4, leading to overestimations of dialyzable FT4 levels. It is unknown whether FT4 overestimates attributable to T4BPs occur. This study reports a sensitive method for detecting and quantifying T4BP cross reactivity in direct dialysis FT4 assays. It is capable of detecting T4BP at a level that is 0.002% of protein levels in normal serum. This T4BP assay was applied to a widely accepted FDA approved direct equilibrium dialysis FT4 method (Nichols). T4BPs without T4 were obtained by removing T4 (ion exchange resin extraction, Amberlite) from a pool of normal serum (normal albumin, TTR, TBG and TT4). After T4 extraction, this serum was dialyzed to equilibrium. When the free T4 RIA used with this equilibrium dialysis FT4 method was applied to this dialysate, there was no displacement of 125I-T4 from anti-T4. When the assay was applied to the retentate containing T4BPs, all 125I-T4 was displaced from anti-T4. A series of solutions with varied T4BP concentrations was produced by serial dilution of the serum retentate with the serum dialysate. The FT4 RIA was applied to each of these dilutions. Progressive T4BP displacements of 125I-T4 from anti-T4 were obtained when serum protein dilutions ranged from 1:10-1:100,000. 125I-T4 binding was reduced by 13%, 57% & 92% at serum protein concentrations that were 0.001%, 0.1% & 1% of the normal serum levels. Comparable 125I-T4 displacements were obtained with FT4 concentrations that were 0.3, 3.0 and 30 ng/dL. Thus, the 125I-T4 FT4 RIA performed as a T4BP assay in the absence of T4. T4BP cross reactivity could be detected, and quantified in terms of T4 binding protein concentrations and/or FT4 overestimations. This is the first application of this T4BP assay to testing T4BP retention in a direct dialysis FT4 assay. There was no T4BP cross reactivity in the assay studied. These data demonstrate that the status of T4BP retention during dialysis and the presence or absence of T4BP cross reactivity in direct dialysis FT4 measurements, can now be based on experimental evidence rather than unsubstantiated assumption.