The purposes of this study were: (1) to clone the cDNA encoding pituitary thyroid-stimulating hormone beta subunit (TSH ) of the Japanese eel, Anguilla japonica, together with its genomic DNA sequence, for phylogenetic analysis, and to study the regulation of the TSH gene expression in cultured pituitaries; and (2) to investigate the transcript levels of pituitary TSH mRNA and the serum thyroxine profiles at different stages of ovarian development before and during silvering in the wild female eels. The maturity of female eels was divided into four stages, juvenile, sub-adult, pre-silver, and silver, based on skin color and oocyte diameter. The genomic DNA of the TSH subunit contains two introns and three exons, and the TSH protein possesses a putative signal peptide of 20 amino acids and a mature peptide of 127 amino acids. The amino acid sequence identities of TSH mature peptide of Japanese eel compared with those of teleosts and other vertebrates are: European eel (98·4%), salmonids (60·6 -61·3%), carps (52·0 -56·7%), sturgeon (48·4%), and tetrapods (42·9 -45·2%). In in vitro studies of the regulation of TSH mRNA it was found that thyrotropin-releasing hormone increased while thyroxine decreased its expression. RT-PCR and real-time quantitative PCR analysis showed that the transcript levels of TSH subunit increased during eel silvering. The serum thyroxine levels also increased in parallel with TSH mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild female Japanese eels.