Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKC. The activation of PKC and PKC coincided with PKD1 activation and while a complex was formed between PKD1 and PKC following aldosterone treatment, there was a concurrent reduction in PKD1 association with PKC. A stable PKD1 knock-down M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of eCFP-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1-suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells , and ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKC /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.