The effects of thyroid hormones on metabolism and development are mediated by thyroid hormone receptors (TRs). We report the cloning and characterization of a TR beta1 cDNA from zebrafish. Southern blot analysis revealed that there is a single genomic locus for the TR beta gene, while the TR alpha gene potentially has two loci. Multiple TR alpha and TR beta transcripts were detected in adult tissues. Using a semiquantitative RT-PCR assay, zygotic expression of TR alpha1 and TR beta1 were shown to occur before the midblastula transition stage. In transiently transfected HeLa cells, TR alpha1 displayed constitutive transactivation in the absence of ligands, which was slightly enhanced by triiodothyronine (T3). The transactivating activity of TR beta1 was strictly ligand-dependent and repressed in the absence of T3. Finally, the T3 induction of TR alpha1 and TR beta1 mRNAs was demonstrated in zebrafish embryos and larvae. The auto-induction of TR alpha1 and TR beta1 may serve a regulatory role during the embryonic and larval development of zebrafish.