A new equilibrium assay for the determination of serum free thyroxine was evaluated in 514 patients. The assay comprises a two-vial-procedure to measure total thyroxine and free thyroxine fraction by use of monoclonal antibodies. Free thyroxine concentrations are calculated from fT4-fraction and total thyroxine concentration readings. In euthyroidism the average free thyroxine fraction (%fT4) was 0.011%, in hyperthyroidism this fraction was elevated, in hypothyroidism it was below normal. In patients with TBG anomalies, TBG values were inversely correlated with fT4 fraction readings. The "euthyroid reference range" of FT4 (SPAC ET) was between 0.70 to 1.78 ng/dl. This euthyroid range of FT4 was determined from TT4 concentrations measured by T4-RIA (SPAC T4 MONO) which were 30% above TT4 values measured by conventional T4-RIA (SPAC T4 POLY; polyclonal antibodies). However, a different euthyroid range of FT4 between 0.55 to 1.30 ng/dl was observed as well as by other investigators when conventional T4-RIA measurements were used for calculation of FT4 values. Our results indicate that calculated FT4 concentration values are highly dependent on the methods used for determination of total thyroxine concentrations. Precision and reproducibility of this two vial equilibrium assay did not meet the requirements mandatory for the application as a clinical routine diagnostic procedure, and its general use for this purpose can as yet not be recommended.