The kidney is a primary target organ of the vitamin D endocrine system, and both vitamin D-deficiency and vitamin D receptor (VDR) ablation lead to impaired renal functions. As an initial step to understand the molecular basis underlying the renal dysfunctions resulted from VDR inactivation, we used DNA microarray technology to search for changes in the gene expression profile in the kidney of VDR knockout mice. Three independent DNA microarray experiments were performed using Affymetrix GeneChips, which included two replicate comparisons between VDR null and wild-type littermates, and a third comparison between 1,25-dihydroxyvitamin D3-treated and vehicle-treated wild-type mice. Based on the assumption that VDR inactivation and vitamin D stimulation cause opposite changes in the expression of vitamin D target genes, we identified 95 genes that displayed the same changes in the two VDR-null/wild-type comparisons but an opposite change in the third assay, of which 28 genes were up-regulated and 67 were down-regulated in VDR null mice. These genes can be divided into several functional categories involved in vitamin D and steroid metabolism, calcium metabolism and signaling, volume and electrolyte homeostasis, signal transduction, transcriptional regulation, cell adhesion, metabolism, immune response, and other functions. These data provide a basis for further investigations into the molecular bases underlying the physiological abnormalities associated with VDR- and vitamin D-deficiency. J. Cell. Biochem. 89: 709–719, 2003. © 2003 Wiley-Liss, Inc.