Rainbow trout ovarian follicles were incubated in vitro with tritiated 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P; maturation-inducing steroid). Within 18–24 h, 56–66% had been converted to tritiated 17α,20β-dihydroxy-4-pregnen-3-one 20-sulfate (identification confirmed by HPLC) and 27% had been taken up (absorbed) by the follicles. Addition of 125 ng of cold (non-tritiated) 17,20β-P to the incubations caused a decrease in the percentage of [3H]-17,20β-P which was sulfated (56% → 10%) and an increase in the percentage that was taken up (27% → 57%). Seven steroids were tested for their effectiveness in decreasing the sulfation and increasing the uptake of tritiated [3H]-17,20β-P. The order of effectiveness was in both cases the same: 17,20β-P > cortisol > 11-deoxycortisol > 17α,20β,21-trihydroxy-4-pregnen-3-one > 17α-hydroxy-4-pregnene-3,20-dione > 17β-estradiol > testosterone. This indicated that the processes of sulfation and uptake of [3H]-17,20β-P were related to each other and led to the hypothesis that, when cold 17,20β-P is added to the medium, it reduces the proportion of [3H]-17,20β-P which is sulfated and thus allows more free [3H]-17,20β-P to enter the ovarian follicles. This hypothesis was supported by the finding that each ovarian follicle had the capacity in vitro to sulfate only ca. 2 ng of [3H]-17,20β-P per 18h but a capacity to take up > 500 ng per 18h.Gonadotropin I, Gonadotropin II, forskolin and phorbol-12-myristate-13-acetate (which all have an affect on steroid biosynthesis) did not affect the amount of 17,20β-P which was sulfated. Sulfating activity was localized in the thecal cell layer of the follicle. The yolk fraction was shown to be responsible for absorbing the [3H]-17,20β-P.