The calcium sensing receptor (CaSR) is a GPCR with a key role in the regulation of calcium homeostasis. Although the initial cloning and characterization of the CaSR in kidney was performed in 1995, discrepancy in the expression pattern within this organ and species differences have been reported by different groups. Determining the expression of the CaSR along the nephron is key to understanding the physiological role of the CaSR in the kidney. We aimed to determine the localisation of the CaSR in rat, human and mouse kidney, using in situ hybridisation and immunohistochemistry methods. In situ hybridization was carried out using a sensitive branched-DNA methodology. CaSR mRNA showed highest expression in the thick ascending limb (TAL), but was also expressed in the distal tubule (DT) and collecting duct (CD), with weaker expression in the proximal tubule (PT). For immunohistochemistry three different antibodies raised against different epitopes of the CaSR were used to determine whether the differences reported previously could be ascribed the different antibodies used. By western blotting, all the three antibodies used produced bands of the expected immunoreactivity and molecular weight for CaSR protein in mouse and rat kidney. Immunolocalisation of the CaSR was similar with the three different antibodies; generally CaSR immunoreactivity was stronger in the medulla and weaker in the cortex. Dual labelling was also carried out using antibodies against specific markers for DT (thiazide-sensitive Na+-Cl- cotransporter, NCC), TAL (Tamm-Horsfall protein) and CD (aquaporin 2), to confirm the exact nephron segments expressing the CaSR. The CaSR co-localized with CD, TAL and DT markers, with the strongest signal in the TAL. Our data show that the CaSR exhibits the strongest expression in the TAL, and that it is also expressed in other nephron segments including the PT, CD and DT. These findings have clarified the expression pattern of the CaSR in kidney in several species, using different techniques. This work will enable the full physiological role of the CaSR in kidney to be determined.