Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of inter-Sertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5–1.2 × 10⁶ cells/cm² on Matrigel-coated dishes or bicameral units for 8–9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β2, and TGF-β3 at different time points were assessed by semiquantitative RT-PCR. In selected experiments, the assembly of inter-Sertoli TJs was monitored by transepithelial electrical resistance measurement. It was found that there was no change in the expression of basic fibroblast growth factor throughout the entire culture period. However, there was a 2-fold reduction in the expression of TGF-β2 and TGF-β3 at the time inter-Sertoli TJs were being assembled. On days 5–8, after the inter-Sertoli TJs had been assembled, the Sertoli cell steady-state messenger RNA levels of TGF-β2 and TGF-β3 increased by as much as 3- and 6-fold, respectively, when compared with Sertoli cells on days 1–3 when TJs were being assembled. Also, it was found that recombinant TGF-β3 added to Sertoli cells cultured in vitro at 1.2 × 10⁶ cells/cm² on Matrigel-coated bicameral units perturbed the inter-Sertoli TJ permeability barrier dose-dependently. Moreover, the presence of TGF-β3 also inhibited the transient and/or basal expression of several TJ-associated proteins, which include occludin, zonula occludens-1, and claudin-11 when inter-Sertoli TJs were being assembled in vitro. These results suggest that TGF-β plays a crucial role in regulating the complicated biochemical events of junction assembly in the testis.