This study was to determine the daily fluctuation of serum thyroxine (tT ₄), free thyroxine (fT ₄ ), 3, 5, 3'-triiodo- thyronine (T ₃)concentrations in healthy dogs.Thyroid function of these dogs was evaluated on the basis of results of TSH response test.Samples for the measurement of serum tT ₄ , fT ₄ , and T₃ concentrations were obtained at 3- hour intervals from 8 :00 to 20 :00.Serum tT ₄ , fT ₄ , and T₃ concentrations were measured by the enzyme chemilu-minescent immunoassay (ECLIA).Mean T ₃ concentrations had no significant differences according to the sample collection time during the day.Mean tT ₄ and fT₄ concentrations at 11 :00 were 3.28 ¡¾0.86 ¥ìg/dl and 1.30 ¡¾ 0.37 ng/dl, respectively and mean tT₄ and fT₄ at 14:00 were 3.54 ¡¾1.15 ¥ìg/dl and 1.35 ¡¾0.12 ng/dl, respectively. These concentrations were significantly high compared with tT₄ and fT₄ concentrations at 8:00, which were 1.75 ¡¾0.75 ¥ìg/dl and 0.97 ¡¾0.25 ng/dl, respectively (p <0.05). According to the sample collection time, mean tT₄ and fT₄concentrations changed with similar fluctuation during the day.Based on these results, it was considered that measurement of serum tT₄ and fT₄ concentrations from 11 :00 to 14 :00 might more easily diagnose the canine hypothyroidism in practice.