Hypothyroidism is associated with impaired urinary diluting ability in humans and animals. The purpose of this study was to examine protein expression of renal aquaporins (AQP) and ion transporters in hypothyroid rats (HT) in response to an oral water load as compared with controls (CTL) and hypothyroid rats replaced with L-thyroxine (HT+T). Hypothyroidism was induced by aminotriazole administration for 10 weeks. Body weight, water intake, urine output, solute and urea excretion, and serum and urine osmolality were comparable among the three groups at the conclusion of the 10-week treatment period. One hour after oral gavage of water 50 ml/kg body weight, HT rats demonstrated significantly less water excretion (8±2 vs. CTL 19±3 vs. HT+T 21±3%, p<0.05), higher minimal urinary osmolality (430±40 vs. CTL 219±60 vs. HT+T 263±30 mOsm/kg H₂O, p<0.05), and decreased serum osmolality (246±6 vs. CTL 266±4 vs. HT+T 267±4 mOsm/kg H₂O, p<0.05) as compared with CTL and HT+T rats. Despite the hypoosmolality, plasma vasopressin concentration was elevated in HT rats (1.17±0.11 vs. CTL 0.71±0.03 vs. HT+T 0.78±0.14 pg/ml, p<0.05). These findings in HT rats were associated with an increase in protein abundance of renal cortex AQP1 (HT 274±11 vs. CTL 100±12 vs. HT+T 143±35 % CTL mean, p<0.01) and inner medulla AQP2 (HT 116±1 vs. CTL 100±1 vs. HT+T 89±2 % CTL mean, p<0.05). AQP3, AQP4, and Na-K-2Cl cotransporter were also increased. Moreover, one hour following the oral water load, HT rats demonstrated a significant increase in the membrane to vesicle fraction of AQP2 by Western blot analysis. The defect in urinary dilution in HT rats was reversed by the V₂ vasopressin antagonist, OPC-31260. In conclusion, impaired urinary dilution in HT rats is primarily compatible with the nonosmotic release of vasopressin and increased protein expression of renal AQP2. The impairment in maximal solute-free water excretion in HT, however, appears also to involve diminished distal fluid delivery.