The purpose of the present study was to modify a previously evaluated enzyme linked immunosorbent assay (Larsson and Lumsden, 1980) for the determination of the thyroxine (T₄) concentration in canine plasma samples. The test kit Enzymun‐Test®^{Boehringer Mannheim} for determination of T₄ concentrations in human plasma was used in connection with the analyzer system Enzymun‐Test®^{Boehringer Mannheim} System ES‐33. The assay protocol originally used by Larsson and Lumsden (1980) was modified by including an additional standard, prolongation of the incubation period for the immunoreaction and the enzymeassay, and reduction of the amount of T₄ conjugate. The intra‐ and interassay coefficient of variations ranged from 3.8% to 12.5%. The detection limit of the assay was 3.9nmol/l which is well below the T₄ concentration observed in plasma from healthy dogs, and the T₄ concentrations determined in plasma from healthy dogs before and 3 hours after administration of TRH (38.0 ± 7.4 nmol/l and 51.8 ± 8.5 nmol/l, respectively) were comparable to those reported in the literature.