In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V₂ receptors (V₂Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V₂Rs have been inconclusive with respect to segment- and cell-type-related V₂R expression. Our study therefore aimed to present a high-resolution analysis of V₂R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V₂R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na⁺-K⁺-2Cl⁻ cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V₂R agonist desmopressin. We found solid expression of V₂R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V₂R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V₂R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V₂R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V₂R expression in macula densa may be related to tubulovascular signal transfer.