Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide (NO). NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. While inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 \x 10⁶ cells/cm² on Matrigel-coated dishes or bicameral units, functional TJ- barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance (TER) across the cell epithelium. The assembly of the TJ-barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ-barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase (sGC) inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ-barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and -tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ-barrier dose-dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G (PKG), was shown to facilitate the Sertoli cell TJ-barrier assembly. Cytokines, such as TGF- and TNF-, known to perturb the Sertoli cell TJ-barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose-dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/sGC/cGMP/PKG signaling pathway.