The description of two or more forms of gonadotropin-releasing hormone (GnRH) in most vertebrates suggests multiple roles for this family of peptide hormones. In order to verify these functions, we analysed the anatomical location, time of initial expression and ontogenic changes in three distinct GnRH receptors (GnRH-Rs) in developing and sexually mature tilapia, using antisera raised against the extracellular loop three of the receptor, which is a determinant in ligand-selectivity and receptor coupling to signalling pathways. In all age groups, including males and females, using in situ hybridization and double-label immunological methods, GnRH-R type IA was colocalized in cells containing luteinizing hormone (LH) beta-subunit in the pituitary. GnRH-R type IB was visualized in prolactin cells and LH cells. The type III GnRH-R was expressed in growth hormone cells. On day 8 after fertilization, GnRH-R type III was first seen in growth hormone cells and, subsequently, on day 15, GnRH-Rs type IA and type IB were first seen in LH and prolactin cells, respectively. On day 25, the receptor occupied area per pituitary and the staining intensity of GnRH-R type IA increased significantly, consistent with the hypothesis that differentiation of GnRH neurones and their inputs to the pituitary coincide precisely with gonadal sex differentiation and steroidogenesis in tilapia. The differential distribution of GnRH-Rs in the pituitary provides the first clear evidence that the three native GnRH variants in tilapia have cognate receptors, each capable of regulating different pituitary endocrine cells.