In mammalian testes Sertoli cells form tight junctions whose function is fundamental for the maintenance of a normal spermatogenesis. Hepatocyte growth factor (HGF) is a cytokine influencing the cellular tight junctions either in normal or in tumor cells. We have previously demonstrated that HGF is expressed in the rat testis and influences many functional activities of somatic and germ cells. We now report that HGF decreases the levels of testicular occludin and influences the position of the molecule in the tight junctions as demonstrated by confocal microscopy analysis. In fact in the presence of the factor occludin was mainly localized in the suprabasal region of the tubules whereas in its absence occludin was prevalently localized in the basal region. Occludin production is known to be regulated by different cytokines including TGFβ. We have investigated the role of HGF in the regulation of the levels of TGFβ and we report that HGF significantly increases the amount of the active fraction of the factor without affecting the amount of the total TGFβ. Urokinase type plasminogen activator (uPA) is closely related with the tight junctions and is one of the molecules able to activate the inactive TGF-β. We found that HGF significantly increases the amount of uPA present in the testis suggesting that HGF regulates the amount of active TGFβ via uPA levels. In conclusion we report that in the testis HGF regulates Sertoli–Sertoli tight junctions inducing a reduction and redistribution of occludin possibly modulating the levels of uPA and active TGFβ. J. Cell. Physiol. 216: 253–260, 2008. © 2008 Wiley-Liss, Inc.