The epithelial Na+ channel (ENaC) mediates Na+ re-absorption in renal cortical collecting ducts. The abundance of ENaC at the cell apical membrane and consequently the rate of Na+ transport are tightly regulated by the adrenal mineralocorticoid hormone aldosterone to maintain whole body Na+ balance. A novel signalling cascade involving protein kinases PKC and PKD has been proposed for rapid aldosterone modulation of ENaC expression at the plasma membrane (1). However, the mechanism and physiological consequences of the rapid sub-cellular re-distribution of ENaC remains to be clearly established. The effect of aldosterone (10 nM) treatment on the rapid trafficking of the ENaCα subunit in polarized M1CCD cells was investigated using immuno-fluorescent staining and confocal microscopy. In untreated cells, ENaCα displayed a punctate cytosolic distribution; while after 5 min aldosterone treatment, ENaCα rapidly localized to the plasma membrane. The signalling cascade responsible for this effect was investigated. Pre-treatment of polarized M1 cells with the mineralocorticoid receptor antagonists eplerenone (1μM) or spironolactone (1μM) inhibited the rapid aldosterone effect on ENaCα trafficking. Pretreatment with Src kinase inhibitor PP2 (100 nM) also prevented aldosterone rapid action on apical localization of ENaCα. PKD1 has been implicated in ENaC trafficking in non-polarized cells (1); we observed that the aldosterone effect was blocked in polarized M1 cells in which PKD1 expression had been suppressed using siRNA. Aldosterone-induced PKD1 activation is dependent on EGFR trans-activation (1), pre-treatment with the EGFR inhibitor AG1478 (5 μM) disrupted the ENaCα sub-cellular re-distribution in polarized M1 cells after treatment with aldosterone. Other steroid hormones such as oestrogen cause EGFR trans-activation via activation of the matrix metalloproteinase (MMP) cascade (2); however the MMP inhibitor GM 6001 (1 μM) did not suppress the rapid aldosterone effect on ENaCα. These data demonstrate that the rapid changes in the sub-cellular localization of ENaCα in polarized M1 cells in response to aldosterone are dependent on cell signalling via MR, Src kinase, EGFR and PKD1 but are independent of MMP cascade activation.