The organic cation transporter-1 (OCT1) mediates the hepatocellular uptake of cationic drugs and endobiotics from sinusoidal blood. The uptake rates of these compounds may depend on OCT1 expression level. Because little is known about the regulation of the human OCT1 (hOCT1) gene, we characterized the hOCT1 promoter with respect to DNA-response elements and their binding factors. By computer analysis, we identified two adjacent putative DNA-response elements for the liver-enriched homodimeric nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) in the hOCT1 promoter. Each element is of the direct repeat (DR)-2 format, containing directly repeated hexamers separated by two bases. In electrophoretic mobility shift assays, both elements directly interacted with HNF-4alpha. A luciferase reporter construct containing the hOCT1 promoter was strongly activated by HNF-4alpha in transiently transfected Huh7 cells. Site-directed mutagenesis of either DR-2 element alone or in combination severely decreased the HNF-4alpha-mediated activation of the hOCT1 promoter, indicating that both elements are functionally important. Because HNF-4alpha is a known target for bile acid-mediated suppression of transcription, we studied whether chenodeoxycholic acid (CDCA) suppresses hOCT1 gene expression by inhibiting HNF-4alpha-mediated transactivation. Treatment of cells with CDCA could indeed suppress the activation of the endogenous hOCT1 gene by HNF-4alpha. In addition, bile acid-inducible transcriptional repressor, small heterodimer partner (SHP), inhibited activation of the reporter-linked hOCT1 promoter and of the endogenous hOCT1 gene by HNF-4alpha. In conclusion, the hOCT1 gene, encoding an important drug transporter in the human liver, is activated by HNF-4alpha and suppressed by bile acids via SHP.