Ligand-binding studies revealed the presence of GnRH-binding sites in African catfish ovary. However, our expression profiling studies failed to detect the previously identified catfish GnRH receptor (cfGnRH-R1) mRNA in this tissue. This negative result instigated us to clone an additional catfish GnRH receptor (cfGnRH-R2) cDNA and study its expression in different tissues in conjunction with the expression of the two catfish GnRH (i.e. cfGnRH and cGnRH-II) genes. The highest cfGnRH-R1 and cfGnRH-R2 mRNA levels were detected in pituitary for cfGnRH-R1 and in brain and ovary for cfGnRH-R2. cfGnRH mRNA was coexpressed with cfGnRH-R1 mRNA in pituitary and brain and with cfGnRH-R2 mRNA in brain and ovary. Ubiquitous expression of cGnRH-II mRNA was observed in all tissues tested, with the highest expression in brain, heart, pituitary, ovary, and head-kidney. Binding studies revealed that cfGnRH-R1 had a higher affinity than cfGnRH-R2 for cGnRH-II, cfGnRH, and various other GnRH agonists. However, this was not reflected in the inositol phosphate or cAMP signal transduction properties of both types of cfGnRH-R. We therefore conclude that in catfish, functional ligand/receptor units evolved by restricted coexpression of a particular receptor in combination with a particular GnRH in particular (nearby) tissue(s).