A new superfusion, two-compartment culture system recently developed in our laboratory was used to investigate the dynamic changes in bidirectional secretion of transferrin (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10(-6) M; follicle stimulating hormone, 0.1 microgram/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to 3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers and for the study of bidirectional secretions under more "physiologic" conditions than those provided by static cultures.