In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT₁ and AT₂. Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor– deficient mice were bred with an Immortomouse®, which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F₂ mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor–deficient cell lines, (AT_1A [−/−], Tag [ /−]), (AT_1B [−/−], Tag [ /−]), and (AT_1A [−/−], AT_1B [−/−], Tag [ / ]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; −3 to 3 μA/cm²), basal monolayer conductance (G, 2 to 10 mS/cm²), Na₃ –phosphate cotransport (ΔIsc of 2 to 3 μA/cm² at 1 mM), and Na₃ –succinate cotransport (ΔIsc of 1 to 9 μA/cm² at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II–stimulated β-arrestin 2 translocation and showed an Ang II–mediated response in wild type but not (AT_1A [−/ −], AT_1B [−/−]) cells. Cell lines were amplified, yielding a virtually unlimited supply of highly differentiated, transport-competent, angiotensin receptor–deficient PCT cell lines.