To further characterize cells of the lower portion of the thin limb of Henle (TLH_lp) under defined conditions in vitro, we developed a technique to enrich this cell population in suspension. TLH_lp cells were isolated by enzymatic digestion of rat inner medulla, elimination of collecting ducts by lectin-coated beads, and differential centrifugation. Immunohistochemical staining of primary cultures of TLH_lp cells with various markers revealed the preparations to be >90% pure. The hormonal stimulation pattern of PGE₂ and cAMP production by arginine vasopressin, angiotensin II, and dopamine in the isolated cells also argued against significant contamination by other cell types. Staining with an antibody against the aquaporin-1 water channel showed the distribution of cells from the ascending and descending limbs to be approximately equal in the isolated population. This technique allows the enrichment of cells from the lower portion of the thin limb of Henle in suspension to a very high degree of purity with the option to start primary cultures. Because these segments of the tubular system in particular are relatively inaccessible for microdissection, the presented method renders the possibility of addressing new questions regarding these tubular segments under defined conditions in vitro.