In this work we studied the effects of the molecular weight (M.W.) of thyroxin (T4)-conjugates and the sample dilution factor on the binding potential (C) of serum T4-binding proteins for these T4-conjugates. We prepared six T4-conjugates with great difference in molecular weight with proteins such as IgG, apoferritin, ferritin, transferrin, and thyroglobulin using p-benzoquinone as bifunctional reagent. The conjugates prepared were characterized in terms of their M.W., the molar ratio of T4 to the carrier protein, and their affinity to bind with the anti-Tr antibody. The binding potential values of serum T4-binding proteins for the T4-conjugates were determined, following appropriate mathematical interpretation of the results, obtained through an assay system containing 125I-labeled conjugated tracers, anti-T4 antibody in great excess compared with the concentration of the tracers, and increasing concentration of T4-binding proteins. We conclude that the M.W. of the conjugates is a parameter which significantly influences the binding of a conjugate to the T4-binding proteins. Additionally, for conjugates of very high M.W. (> 650,000), zero C values were obtained using 20-fold sample dilution in the final incubation mixture.