We have analyzed the functional and morphological effects of corticosteroid hormones in a SV40-transformed rabbit cortical-ascending-limb (CAL) cell line (RC.SV2, Vandewalle et al., 1989) having mineralocorticoid (MR) and glucocorticoid (GR) receptors (Rafestin-Oblin et al., 1993). Both aldosterone and dexamethasone (5 x 10(-8) M) induced a marked increase in (3H)ouabain binding (used to quantify membrane Na(+)-K+ ATPase) detectable as early as 6 hours and maximal at 24 hours (+56-57%) (due to a 1.6-1.8-fold increase in cell membrane binding sites without Kd alteration), and significantly augmented the ouabain-sensitive component of Rb+ influx. Triiodothyronine (T3, 10(-9) M) also stimulated ouabain binding by 21% but was not permissive for steroid action, whereas 5 micrograms/ml insulin had no effect. Both steroid hormones, T3 and insulin induced the formation of domes that was tightly correlated with ouabain binding (r = 0.949) except for insulin. The effects of aldosterone and dexamethasone on cell monolayers and cell ultrastructure were, however, strikingly different as aldosterone induced a marked amplification of basolateral areas with appearance of large intercellular spaces, reminiscent of the changes observed in deoxycorticosterone-treated rats, whereas dexamethasone predominantly influenced cell height. This discrepancy might be due to specific occupancy of MR and GR by aldosterone and dexamethasone, respectively, and/or to nongenomic effects of dexamethasone. We have thus characterized a cell culture model making it possible to analyze the actions of mineralocorticoid and glucocorticoid hormones in the mammalian kidney.