Abstract In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the α-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of βB-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the βB-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of βB-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the βB-subunit of inhibin changed markedly. The meaning of this changing ratio between βB-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of βB-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4β-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the βB-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin αβ-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the α-subunit was increased. It is concluded that the level of the βB-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels.