Follistatin is a single-chain glycoprotein initially identified in the mammalian ovary. As a specific binding protein of activin, it effectively modifies the paracrine/autocrine roles of activin in a variety of tissues including the ovary. In the zebrafish, we have demonstrated that the human chorionic gonadotropin (hCG)-induced oocyte maturation and oocyte maturational competence can be blocked by follistatin, suggesting a role for ovarian activin in the signaling pathway of gonadotropin in the zebrafish ovary. The up-regulation of zebrafish ovarian activin betaA subunit by gonadotropin further supports this hypothesis. Since follistatin has extremely high affinity for activin, its expression level in various tissues is critical in fine-tuning local activin activities. In the present study, we investigated the regulation of follistatin expression by gonadotropin in a primary culture of zebrafish ovarian follicle cells using semi-quantitative RT-PCR. Both hCG and goldfish pituitary extract strongly increased the expression of follistatin in the cultured follicle cells in clear time- and dose-dependant manners. The effect of hCG (15IU/ml) reached the maximal level at 2h of treatment and longer treatment (4-8h) led to decreased response. The up-regulation of follistatin expression by hCG could be mimicked by all the drugs that increase the intracellular cAMP level including 8-Br-cAMP, db-cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). The hCG (15IU/ml)- and forskolin (10microM)-induced follistatin expression could be blocked by H89 (10microM), a specific protein kinase A (PKA) inhibitor. These results strongly suggest that the regulation of follistatin expression in the zebrafish ovary by gonadotropin is primarily mediated by cAMP-PKA signaling pathway. The up-regulation of follistatin mRNA by gonadotropin in cultured zebrafish ovarian follicle cells, together with our previous studies on gonadotropin regulation of activin beta subunits, suggests that the ovarian activin-follistatin system is tightly controlled by gonadotropin in fish ovary.