In the Japanese eel Anguilla japonica, the administration of exogenous GTH is necessary for the artificial induction and completion of gonadal maturation due to its GTH deficiency under captive conditions. The isolation of native eel GTH has not been accomplished, which has made it difficult to fully elucidate the biological functioning of the two GTHs (FSH and LH) in eel. In this study, we attempted to produce a recombinant Japanese eel GTH (rjeFSH) having biological activity using methylotrophic yeast, Pichia pastoris in order to gain more understanding of the functioning of GTH in this species. An expression vector in which jeFSHbeta and GTHalpha subunit cDNAs were tandemly connected was constructed. P. pastoris was transformed with the vector, and rjeFSH was expressed. The rjeFSH thus expressed was detected by Western blot analysis. The glycoprotein fraction of the yeast culture supernatant was separated by native PAGE, and a band showed positive reaction with anti-GTHalpha and FSHbeta antisera similarly, suggesting that both subunits are associated. After deglycosylation, both subunits were decreased in molecular mass, indicating that rjeFSH was glycosylated. In in vitro assay, rjeFSH stimulated the release of testosterone and 11-ketotestosterone from immature eel testis, whereas release was not stimulated in maturing eel testis. This is the first report investigating the biological activity of eel GTH using the recombinant eel FSH.