In most teleosts, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) serves as the most potent maturation-inducing steroid (MIS) to initiate final oocyte maturation. The maturational competence or the responsiveness of oocytes to DHP increases when the ovarian follicles approach the final stage of growth. In the zebrafish, we demonstrated in the present study that full-grown oocytes (∼0.7 mm) exhibited the highest maturational competence, which diminished progressively with decreasing size of the follicles. Using midvitellogenic follicles (0.49–0.56 mm), which had little response to DHP, as the material, the present study aimed at investigating the endocrine and paracrine mechanisms that regulate maturational competence of the oocytes. In agreement with the results of studies in other teleost fish, pretreatment of follicles with gonadotropin (hCG) significantly enhanced the responsiveness of midvitellogenic oocytes to DHP in a clear time- and dose-dependent manner. Interestingly, activin, an ovarian growth factor, also had a potent stimulatory effect on the acquisition of oocyte maturational competence. Pretreatment with either recombinant human activin A or goldfish activin B significantly increased the rate of DHP-induced oocyte maturation from ∼3% to ∼70%, also in a clear dose-dependent manner. Similar to the results with hCG, pretreatment with activin alone had no effect in inducing maturation of midvitellogenic oocytes without subsequent DHP treatment, although both exhibited a strong effect in promoting maturation of full-grown oocytes. The effect of activin on maturational competence of oocytes could be reduced by cotreatment with follistatin, a potent activin-binding protein. Interestingly, follistatin treatment also significantly reduced the effect of hCG on maturational competence of oocytes, suggesting a mediating role for endogenous activin or activin-related molecules in the action of gonadotropin. The effects of hCG and activin on maturational competence of oocytes could be significantly inhibited by actinomycin D (1 μg/ml) and completely blocked by cycloheximide (1 μg/ml), suggesting that the hCG and activin-induced acquisition of oocyte maturational competence involves de novo protein synthesis at both the transcriptional and translational levels.