The mouse octn transporter family consists of three genes, octn1, octn2 and octn3. The gene products octn2 and octn3, which transport carnitine with high affinity, are both expressed in testis, where carnitine is required to maintain sperm cell motility. Here, we focused on the regulatory mechanism of the expression of octn3 in an attempt to determine whether the differential tissue expression profiles of the octn2 and octn3 genes reflect distinct physiological roles of octn2 and octn3. The promoter activity of the octn3 gene was examined by luciferase assay and gel mobility shift assay using the mouse Sertoli cell line TM4 as host cells. Deletion-mutant assay demonstrated that a gene segment of the 5'-untranslated region located at about -500 bp relative to the transcription start site is required for constitutive octn3 transcription. Deletion of the Sp1-binding site within the region resulted in loss of transcriptional activity. In addition, overexpression of Sp1 in TM4 cells led to a further increase of transcription of octn3. These results demonstrated that Sp1-binding sites are necessary and sufficient for constitutive octn3 gene transcription. Furthermore, the expressions of both of octn2 and octn3 genes in TM4 cells were up-regulated by palmitic acid, whereas carnitine increased only the expression of octn2 without any change in octn3 expression. Accordingly, the expressions of octn2 and 3 are regulated by distinct mechanisms, suggesting distinct roles of octn2 and octn3 in carnitine transport.