The analytical and clinical performance of three radioisotopic methods for the measurement of free thyroxine-Gamma Coat (125I) Free T4, RIA-gnost FT4 and Amerlex-MAB* FT4-were assessed in comparison with the Scalvo two-step chromatographic method. The analytical evaluation indicated excellent performance of Amerlex-MAB FT4. For the other methods, precision and sensitivity were comparable and acceptable for routine use. Only Gamma Coat FT4 showed a significant positive intra-assay drift. None of the methods showed a significant correlation with thyroxine-binding globulin. Amerlex-MAB FT4 results were weakly positively correlated with albumin. Sclavo, RIA-gnost and particularly GammaCoat FT4 results were affected by the presence of increased concentrations of free fatty acids. All methods classified hyperthyroid patients correctly. Slight overlaps existed between the hypothyroid and euthyroid populations. Significant decreases in free thyroxine (FT4) during the third trimester of pregnancy were detected by all assays. In patients with chronic renal failure, serum FT4 was within reference limits more often when measured by Sclavo or Amerlex-MAB methods than by RIA-gnost or GammaCoat techniques. In conclusion, all assays performed well from both technical and diagnostic points of view