Throughout the mammalian spermatogenic pathway, differentiating spermatogenic cells remain in close contact with somatic Sertoli cells, and this has been considered to be essential for the proliferation, differentiation, and survival of spermatogenic cells. It is thought that Sertoli cells form tight junctions to protect developing spermatogenic cells against harmful agents and provide several nutrients for spermatogenesis from the blood stream. Accordingly, Sertoli cells should regulate the movement of various nutritious and xenobiotic compounds by selective membrane transporters. However, the information on membrane transporters in Sertoli cells is limited. In the present study, we characterized the transport systems of organic cations in Sertoli cells. Uptake of [14C]tetraethylammonium (TEA) was measured by primary-cultured rat Sertoli cells. Initial uptake of TEA was concentration dependent, and an Eadie-Hofstee plot indicated the involvement of two saturable transport systems. The apparent Km values of high- and low-affinity components were comparable to those of previously known organic cation transporter (OCT) or organic cation/carnitine transporter (OCTN). In addition, OCT1, OCT3, OCTN1, and OCTN2 were expressed in Sertoli cells. In conclusion, multiple organic cation transporters, OCTs and OCTNs are expressed in Sertoli cells and the cells regulate transport of cationic compounds to protect and/or maintain the spermatogenesis in testis as the blood-testis barrier.