Immunosuppressive agents such as FK506 and rapamycin inhibit aldosterone stimulated Na⁺ transport in A6 cells. Concentration dependence is consistent with the known affinities of these agents for immunophillins. The inhibition was also dependent on time, requiring preincubation with FK506 or rapamycin before inhibition was seen. The present studies were designed to determine whether this inhibition was pretranscriptional, and whether it was due to an effect on either receptor translocation or nuclear accumulation. Since transport effects of steroids in A6 cells are mediated by glucocorticoid receptors (GR), we examined to transcriptional response of GR-regulated reporters transfected into these cells. Pre-incubation of cells with FK506 and rapamycin completely blocked reporter gene activation, while preincubation with cyclocporin A partially inhibited this activation. A minimum of 8 hours pre-incubation was required before the effect was seen. Using a transiently transfected GFP-GR construct, we examined the effect of FK506 and rapamycin on GR translocation. GR translocation induced by Dexamethasone was extremely rapid (< 5 min) and was largely unaffected by FK506 or rapamycin but completely blocked by geldanamycin. Digital deconvolutions revealed a punctate nuclear accumulation of GR which was still seen following preincubation with immunosuppressive agents. These agents clearly inhibit steroid action by blocking GR-stimulated gene transcription, but this effect is not mediated by altered translocation or nuclear accumulation of receptors. Inhibition of steroid regulated gene transcription by immunosuppressive agents may explain the electrolyte abnormalities seen in patients receiving these drugs.