Aquaporins play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial and other tissues. Aquaporin proteins are abundantly expressed in the mammalian kidney where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal aquaporins have been carried out in laboratory rodents and sheep; no data have been published on the expression of aquaporins in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots confirmed high expression of AQP1-4 in the equine kidney. These observations confirm that aquaporins are expressed in the equine kidney and are found in similar nephron locations to mouse, rat and human kidney. Equine renal aquaporin proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxaemia.