The regulation of the OSTα and OSTβ expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue slices. The gradient of protein and mRNA expression in segments of the intestine for rOSTα and rOSTβ paralleled that of rASBT. OSTα and OSTβ mRNA expression, quantified by qRT-PCR, in rat jejunum, ileum, colon and liver, and in human ileum and liver was positively regulated by FXR and GR ligands. In contrast, the VDR ligand, 1,25(OH)2D3 decreased the expression of rOSTα-rOSTβ in rat intestine, but had no effect on human ileum, and rat and human liver slices. Lithocholic acid (LCA) decreased the expression of rOSTα and rOSTβ in rat ileum but induced OSTα-OSTβ expression in rat liver slices, and human ileum and liver slices. The PXR ligand, pregnenolone-16α carbonitrile (PCN) had no effect. This study suggest that, apart from FXR ligands, the OSTα and OSTβ genes are also regulated by VDR and GR ligands and not by PXR ligands. This study show that VDR ligands exerted different effects on OSTα-OSTβ in the rat and human intestine and liver compared with other nuclear receptors, FXR, PXR, and GR, pointing to species- and organ-specific differences in the regulation of OSTα-OSTβ genes. Copyright © 2009 John Wiley & Sons, Ltd.