Yolk-free follicle cells during final meiotic maturation were incubated with a radioactively labeled steroid precursor, and the maturation-inducing activity of the steroid metabolites produced was tested in an in vitro assay based on GVBD. Of the metabolites obtained by TLC, whose purity and final characterization were confirmed by recrystallization to constant specific activity, two steroids, 17,20β-P and 20β-S, exhibited maturation-inducing activity in vitro. However, 20β-S was overwhelmingly more effective and faster at inducing GVBD in vitro than 17,20β-P. During final oocyte maturation in the tiger puffer, a shift in steroidogenic enzymes, a decrease in C17,20-lyase activity, and an increase in 20β-HSD activity, were found, as previously reported in salmonids and medaka. In addition to this shift in steroidogenic enzyme activity, however, high and increasing 21-hydroxylase activity throughout all the oocyte developmental stages was a distinctive feature in tiger puffer follicles. This 21-hydroxylase activity likely enables ovarian follicles to accumulate enough 11-deoxycortisol during vitellogenesis for 20β-S production on a massive scale during GVBD. Thus, this study provides not only evidence on the physiological role of 20β-S as a MIH in the tiger puffer, but also fact on enzymatic kinetics in 20β-S biosynthesis.