Recent studies in our laboratory have shown that in some teleosts, 3,5-di-iodothyronine (T₂ or 3,5-T₂) is as bioactive as 3,5,3′-tri-iodothyronine (T₃) and that its effects are in part mediated by a TRβ1 (THRB) isoform that contains a 9-amino acid insert in its ligand-binding domain (long TRβ1 (L-TRβ1)), whereas T₃ binds preferentially to a short TRβ1 (S-TRβ1) isoform that lacks this insert. To further understand the functional relevance of T₂ bioactivity and its mechanism of action, we used in vivo and ex vivo (organotypic liver cultures) approaches and analyzed whether T₃ and T₂ differentially regulate the S-TRβ1 and L-TRβ1s during a physiological demand such as growth. In vivo, T₃ and T₂ treatment induced body weight gain in tilapia. The expression of L-TRβ1 and S-TRβ1 was specifically regulated by T₂ and T₃ respectively both in vivo and ex vivo. The TR antagonist 1–850 effectively blocked thyroid hormone-dependent gene expression; however, T₃ or T₂ reversed 1–850 effects only on S-TRβ1 or L-TRβ1 expression, respectively. Together, our results support the notion that both T₃ and T₂ participate in the growth process; however, their effects are mediated by different, specific TRβ1 isoforms.