It has long been known that Na⁺ channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na⁺ channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional ( and ), novel (, , and ), and atypical (, , and ) groups. Steady-state stimulation of Na⁺ transport with aldosterone was accompanied by a specific decrease of PKC protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na⁺ transport, also decreased PKC levels. These effects were additive, indicating separate mechanisms that converge at the level of PKC. These effects were not accompanied by changes of PKC mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na⁺ transport.