Two genomic contigs of putative growth hormone receptors (GHRs) were identified in fugu and zebrafish genomes by in silico analysis, suggesting the presence of two GHR subtypes in a single teleost species. We have tested this hypothesis by cloning the full-length cDNA sequence of a second GHR subtype from the black seabream in which the first GHR subtype had been previously reported by us. In addition, we had also cloned the sequences of both GHR subtypes from two other fish species, namely the Southern catfish and the Nile tilapia. Phylogenetic analysis of known GHR sequences from various vertebrates revealed that fish GHRs cluster into two distinct clades, viz. GHR1 and GHR2. One clade (GHR1), containing 6 to 7 extracellular cysteine residues, is structurally more akin to the non-teleost GHRs. The other clade (GHR2), containing only 4 to 5 extracellular cysteine residues, is unique to teleosts and is structurally more divergent from the non-teleost GHRs. In addition, we had examined the biological activities of both GHR subtypes from seabream using a number of reporter transcription assays in cultured eukaryotic cells and demonstrated that both of them were able to activate the Spi 2.1 and beta-casein promoters upon receptor stimulation in a ligand specific manner. In contrast, only GHR1 but not GHR2 in seabream could trigger the c-fos promoter activity, indicating that the two GHR subtypes possess some differences in their signal transduction mechanisms. Also, the expression of GHR2 is significantly higher than GHR1 in many tissues of the seabream including the gonad, kidney, muscle, pituitary and spleen. In vivo hormone treatment data indicated that cortisol upregulated hepatic GHR1 expression in seabream but not GHR2, whereas testosterone decreased hepatic GHR2 expression but not GHR1. On the other hand, hepatic expression of both GHR1 and GHR2 in seabream was decreased by estradiol treatment.