Renal tubular NHE3, the Na+/H+ exchanger, is a critical enzyme for electrolyte and acid-base homeostasis in the kidney. We previously demonstrated that the expression of this gene in the kidney followed a circadian rhythm directly regulated by clock genes acting on E-box elements present on its promoter region. In the present study, we further characterize the circadian expression of NHE3 in the mice kidney by in situ hybridization, and refine quantification of gene expression using real-time PCR combined with laser capture micro-dissection. We show NHE3 mRNA was strongly expressed in the inner stripe of the outer medulla and weakly in the cortex. Further realtime PCR data from dissected medullary nephron demonstrated clear circadian oscillations in the thick ascending limbs and the thin descending limbs, but not in the collecting ducts. The circadian changes of this molecule in the renal medulla may partially contribute to the circadian change of urinary electrolyte secretion.