Three distinct thyroid hormone receptor (TR) cDNAs (gTRalphaA, gTRalphaB and gTRbeta) were cloned and sequenced in orange-spotted grouper (Epinephelus coioides). The cDNA of gTRalphaA consisted of 1555-bp and encoded a putative protein of 416 amino acids, while the cDNA of gTRalphaB consisted of 1459-bp and encoded 398 amino acids. The cDNA of gTRbeta is 1470-bp in length and encoded 395 amino acids. The cDNAs of gTRalphaA and gTRalphaB had 68.7% identity in nucleotide sequence and 83.9% identity in deduced amino acid sequence, and shared 57.8%, 57.9% identity in nucleotide sequence and 76.5%, 76.6% in deduced amino acid sequence with gTRbeta, respectively. mRNA expression of gTR was determined by using real-time RT-PCR. The zygotic mRNA expression of gTRalphaA, gTRalphaB and gTRbeta was already presented at the blastula stage, and decreased by gastrulation and body segment appearance. Both gTRalphaA and gTRbeta mRNAs dramatically increased by brain vesicle appearance and lens formation stages, whereas the magnitude of the increase of gTRbeta was higher than that of gTRalphaA. However, gTRalphaB remained relatively constant. All three gTR mRNAs were detected in various tissues of adult fish, with considerable levels found in the pituitary and brain. The expression of gTRalphaA and gTRalphaB was ubiquitous, while the gTRbeta transcription in the pituitary was higher than other tissues. The results suggest that gTRs may be important in adult grouper brain function and that gTRalphaA and gTRalphaB may be important factors in metabolic regulation. In addition, gTRbeta may be the major TR isoform in the regulation of TSH activity by TH feedback in grouper.