There have been, and continue to be, reports of discordant free thyroxine (T 4 ) 3 measurements when different free-T 4 methods are applied to the same serum samples (1-4 ). Our laboratory has attempted to identify some of the factors that contribute to these disparities.There are 4 separate components of serum T 4 : free T 4 , albumin-bound T 4 , transthyretin-bound T 4 , and thyroxine-binding globulin (TBG)-bound T 4 . Free T 4 is the one component that moves across semipermeable membranes during equilibrium dialysis and ultrafiltration. As Midgley and Christofides point out, the gold standard free-T 4 methods are designed to measure this component.We applied analog-type methods and an equilibrium-dialysis/immunoassay gold standard method to carefully prepared solutions in which we varied these 4 components of serum T 4 . This equilibrium-dialysis method agrees closely with a liquid chromatographytandem mass spectrometry method applied to serum ultrafiltrates (4 ). We also applied this equilibrium dialysis/immunoassay and a group of 7 analog-type free-T 4 assays to solutions of T 4 that did not include T 4 -binding proteins (5 ); that is, the only form of T 4 was free T 4 . The analog-type assays required 28-to 588-fold higher concentrations of free T 4 than the equilibrium dialysis/immunoassay to generate similar assay results. These results highlight the widely differing characteristics of the antibodies used in these assays. Midgley and Christofides state that analog-type methods are not suitable for application to "pure" analyte solutions. We understand that they are not suitable for the measurement of free T 4 in the absence of protein-bound T 4 ; however, we are not persuaded that applying any assay to the substance it reports is inappropriate, when the goal is to identify the factors that influence assay results.When albumin, transthyretin, and TBG are held constant and the total T 4 concentration is varied, the concentrations of free T 4 , albumin-bound T 4 , transthyretin-bound T 4 , TBG-bound T 4 , and total T 4 are aligned with one another. That is, they are proportionate covariables. In a recent experiment, the total T 4 concentration was varied while albumin, transthyretin, and TBG concentrations were within the reference interval and constant. Seven analog-type free-T 4 assays were applied to these T 4 solutions. Analog-type free-T 4 estimates correlated closely with dialyzable T 4 concentrations (Fig. 1). This result prompts a question: What happens to analog-type free-T 4 estimates when these 5 T 4 components are not aligned?In a previous study, albumin, transthyretin, TBG, and total-T 4 concentrations were proportionate covariables, and dialyzable T 4 was held constant (6 ). Five analog-type free-T 4 assays were applied. Free-T 4 estimates obtained with analog-type assays reflected the concentrations of T 4 -binding proteins and total T 4 . They did not reflect the fixed dialyzable T 4 concentration. These data are direct evidence that total T 4 and/or T 4 -binding proteins influence such as...