Human organic cation transporter 1 (OCT1; SLC22A1) plays important roles in the hepatic uptake of cationic drugs. The functional characteristics of this transporter have been well evaluated, but molecular information regarding transcriptional regulation is limited. In the present study, therefore, we examined the gene regulation of OCT1 gene focusing on basal core expression. An approximately 2.5-kb fragment of the OCT1 promoter region was isolated and promoter activity was measured by luciferase assay in the human liver cell lines Huh7 and HepG2. Deletion analysis suggested that the region spanning -141/-69 was essential for the basal core transcriptional activity, and that this region contained the sequence of a cognate E-box (CACGTG). The E-box is known to be bound by the basal transcription factors upstream stimulating factors (USFs), and the functional involvements of USF1 and USF2 were confirmed by a trans-activation effect, a mutational analysis of the E-box, and an electrophoretic mobility shift assay. The trans-activation effect of USFs on the OCT1 promoter was further stimulated by hepatocyte nuclear factor 4, a liver-enriched transcription factor. There were no polymorphisms in the proximal promoter region (about 400 bp) of OCT1 gene (n=109). These findings indicated that both USF1 and USF2 bind to an E-box sequence located in the OCT1 core promoter region and are required for the basal gene expression of this transporter.