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Abstract: Recombinant gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were previously produced by baculovirus in silkworm larvae Bombyx mori. These GTHs are heterodimeric hormones consisting of a common α subunit and an FSHβ or LHβ subunit; the GTH subunits were produced separately in silkworm larvae. In order to simplify the production process of the hormones and increase production efficiency, we produced single-chain FSH (scFSH) and LH (scLH) in which the FSHβ or LHβ subunit was fused to the α subunit. Biological activities of these single-chain GTHs produced by baculovirus–silkworm larvae system were analyzed in vivo using male goldfish, female bitterling Rhodeus ocellatus ocellatus, and male Japanese eel Anguilla japonica. Injection of hemolymph containing scFSH or scLH induced milt production in male goldfish. scLH induced ovulation in bitterling but scFSH showed lower potency. Both scLH and scFSH induced the initiation of spermatogenesis in the testes of sexually immature male eels after 5–6 administrations of the recombinant GTHs at seven day intervals. Spermatozoa were observed in scLH-administered eels and spermatids in scFSH-administered eels whereas the testes of eels administered with hemolymph of untreated silkworm larvae remained at the spermatogonia stage. Thus, single-chain GTHs of goldfish produced by baculovirus in silkworm larvae showed in vivo biological potency not only in the final phase of gametogenesis (milt production and ovulation) but also in the early phase of gametogenesis (spermatogenesis). We suggest that single-chain fish GTHs could be used for the induction of gonadal development in aquaculture fishes. [Copyright &y& Elsevier]