The presence of mineralocorticoid (MR) and glucocorticoid (GR) receptors was investigated in two renal tubular cell lines, derived from primary cultures of isolated rabbit kidney cortical cells infected with the wild-type SV40 virus, which exhibit thick ascending limb (RC.SV2) and collecting tubule (RC.SV3) phenotypes (Vandewalle et al. J. Cell. Physiol. 141, 1989, 203-221). MR and GR were quantified, in cell monolayers and cell cytosolic fractions, with [3H]aldosterone, [3H]dexamethasone and [3H]RU486, an antiglucocorticoid with no affinity for MR. Cytosolic receptors from RC.SV2 and RC.SV3 cells labeled with [3H]aldosterone, [3H]dexamethasone or [3H]RU486 sedimented at approximately 8 S in a 15-40% glycerol gradient. All steroids displaced bound [3H]dexamethasone to the same extent, suggesting that dexamethasone bound to both MR and GR: under the conditions of assay, [3H]aldosterone binds exclusively to MR, and [3H]RU486 to GR. In both RC.SV2 and RC.SV3 cells, [3H]aldosterone bound to one class of high affinity sites (Kd 0.14-0.8 nM; Nmax 8 to 22 fmol/mg protein). In both cell lines, the number of high affinity binding sites for [3H]dexamethasone ranged from 9 to 18 fmol/mg protein with an affinity of 0.5-1.3 nM. Compared to renal cortex, the most striking observation was a marked decrease in [3H]dexamethasone binding in primary cultures and SV40-transformed cells. These results indicate that MR and GR are expressed in two established mammalian kidney tubular cell lines providing new models of cultured renal cells for studies on the physiological effects of corticosteroid hormones.