A procedure was developed for isolating thick ascending limb cells from either the outer medulla or the inner cortex from rabbit kidneys. Dispersed cells derived from the medulla or cortex were incubated with goat anti-human uromucoid (Tamm-Horsfall glycoprotein) serum, washed, and applied to culture dishes coated with affinity-purified anti-goat immunoglobulin G. Nonadherent cells were removed by washing. Routinely, 10(6) or 7 X 10(4) adherent cells were obtained per gram of rabbit outer medulla or inner cortex, respectively. Greater than 97% of the adherent cells stained for Tamm-Horsfall antigen, and examination of freshly isolated cells by transmission electron microscopy established that they had morphological properties expected for thick limb cells. Freshly isolated medullary thick limb (MTALH) cells consistently accumulated cAMP in response to arginine vasopressin (AVP), thyrocalcitonin, prostaglandin E2 (PGE2), and glucagon. PGE2, thyrocalcitonin, parathyroid hormone, and AVP, but not isoproterenol or glucagon, reproducibly stimulated cAMP accumulation in freshly isolated cortical thick limb (CTALH) cells. MTALH cells produced immunoreactive PGE2 when incubated with 10 microM arachidonic acid. In summary, large numbers of highly purified and hormonally responsive rabbit MTALH and CTALH cells can be obtained by immunodissection using commercially available antibody preparations. Because the Tamm-Horsfall antigen is present as an extracellular determinant on thick ascending limb epithelia from many species, this general approach likely can be used to isolate CTALH and MTALH cells from most mammalian kidneys.