In lithium-induced nephrogenic diabetes insipidus (NDI), alteration in renal medullary osmolyte concentrations has been assumed but never investigated. Amiloride can modify lithium-induced NDI but the impact of amiloride in lithium-induced NDI on renal medullary osmolytes, aquaporins and urea transporters is unknown and is the basis of this study. Rats fed lithium (60 mmol kg dry food^-1) over four weeks developed NDI. Urine osmolality fell to 287 ± 19 mOsm kg^-1 (controls 1211 ± 90 mOsm kg^-1). Organic osmolytes in the renal medulla showed significant decreases compared with controls (inositol 221 ± 35 to 85 ± 10 mmol kg^-1 protein; sorbitol 35 ± 9 to 3 ± 1 mmol kg^-1 protein; glycerophosphorylcholine (GPC) 352 ± 80 to 91 ± 20 mmol kg^-1 protein; and glycine betaine 69 ± 11 to 38 ± 38 mmol kg^-1 protein). Medullary urea content fell from 2868 ± 624 to 480 ± 117 mmol kg^-1 protein. Concurrent administration of amiloride (0.2 mmol l^-1) in the drinking water restored urine osmolality (1132 ± 154 mOsm kg^-1), and reduced urine volume. Medullary osmolyte content were restored to control values (inositol, 232 ± 12; sorbitol 32 ± 6; GPC, 244 ± 26; glycine betaine, 84 ± 5 mmol kg^-1 protein). Medullary urea rose to 2122 ± 305 mmol kg^-1 protein. Reduced aquaporin 2, aquaporin 3, and urea transporter (UT-A1) expression was significantly reversed following amiloride therapy. Data presented here provide further understanding of how amiloride may substantially restore the lithium-induced impaired renal concentrating mechanism.