Throughout spermatogenesis, the Sertoli cell blood-testis barrier (BTB) is strictly regulated by cytokines, which mediate its timely restructuring, thereby allowing spermatocytes to enter the adluminal compartment of the seminiferous epithelium for development into spermatozoa. The aim herein was to investigate whether germ cells play a role in BTB restructuring via the action of interleukin-1α (IL-1α) since germ cells are known to control Sertoli cell production of this cytokine, and if yes, how these effects are mediated. When Sertoli cells were isolated from Sprague-Dawley rats and plated at high density, IL-1α (100 pg/ml) was shown to "open" the Sertoli cell barrier when its integrity was assessed by transepithelial electrical resistance measurements. Further investigation of Sertoli cells treated with IL-1α revealed striking changes in the cellular distribution of actin filaments when compared to untreated cells. These effects at the Sertoli cell barrier were mediated, in part, by epidermal growth factor receptor pathway substrate 8 (Eps8; an actin bundling and barbed-end capping protein) and actin-related protein 3 (Arp3; a component of the actin nucleation machinery). As important, an increase in the kinetics of occludin internalization but a decrease in its rate of degradation was noted following IL-1α treatment. These results indicate that IL-1α is a critical regulator of BTB dynamics.