We incubated different radiolabeled steroid precursors with intact chub mackerel ovarian follicles to clarify the synthetic pathways of steroid hormones during vitellogenesis and following final oocyte maturation (FOM). During vitellogenesis, estradiol-17β (E2) was synthesized from pregnenolone via 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The physiological significance of the intermediate metabolites of E2 in the ovarian follicles was examined by comparing follicular steroidogenesis between gonochoric and hermaphroditic fish species. After vitellogenesis, the steroidogenic pathway shifted from E2 to maturation-inducing hormone (MIH) production owing to the inactivation of 17,20-lyase and the activation of 20β-hydroxysteroid dehydrogenase. Of the new steroids produced during FOM, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was most effective at inducing germinal vesicle breakdown in vitro. Circulating levels of 17,20β-P increased specifically around the time of germinal vesicle migration, while another FOM-specific 20β-hydroxylated progestin, 17,20β,21-trihydroxy-4-pregnen-3-one, was present at consistently low levels during FOM. These results indicate that 17,20β-P is the MIH of chub mackerel.