In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB, or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([³H]guanine) and pyrimidine ([³H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time- and concentration-dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single sodium-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases, but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.