Two types of gonadotropin-releasing hormone (GnRH) receptors were found in the pituitary of tilapia (t), named GnRHR type 3 (tGnRHR3) and GnRHR type 1, according to phylogenetic analysis. tGnRHR3 is highly expressed in the posterior part of the pituitary which contains LH and FSH cells. We characterized tGnRHR3 in terms of both LH release rate and receptor internalization rate in response to continuous exposure to GnRH. Constant exposure of tilapia pituitary fragments to salmon GnRH analog (sGnRHa) resulted in an increased secretion rate for 3 h, followed by a gradual decline, taking 17-19 h, to the basal secretion rate. A chimera between tGnRHR3 and green fluorescent protein (GFP) was created and used to observe the changes in receptor distribution and translocation, activated by agonist with time. The results suggested that the receptor is initially localized at the plasma membrane and upon activation by a homologous ligand (e.g. sGnRHa) undergoes relatively rapid endocytosis. In summary, the present work demonstrates that tGnRHR3 has already undergone endocytosis after 30 min, while desensitization of LH release occurs only after 17-19 h. It is concluded that for tGnRHR3, internalization of the receptor is not exclusively responsible for the desensitization of LH release.