The A663T polymorphism of the -subunit of the human Epithelial Sodium Channel (hENaC) increases the functional and surface expression of -hENaC in Xenopus oocytes, and the context of this residue in the C-terminus of -hENaC is important for this effect. Query of a phosphoprotein database suggested that the -T663 residue of hENaC might be a substrate for phosphorylation by Casein Kinase 1 (CK1). We tested the hypotheses that phosphorylation of -T663-hENaC by CK1 would regulate the increased functional and surface expression of -T663-hENaC vs. -A663-hENaC in oocytes. General inhibition of CK1 with IC261 decreased the functional and surface expression of -T663-hENaC, but not -A663-hENaC. This decrease in -T663-hENaC functional expression resulted from reduced delivery of -T663-hENaC to the oocyte membrane. IC261 also inhibited the functional expression of -T692-mENaC and a chimeric m(1-678)/h(650-669)-T663, m ENaC, but not -A692-mENaC or m(1-678)/h(650-669)-A663, m ENaC. These data suggest that additional residues outside of the -hENaC C-terminus are important for modulation of -T663-hENaC trafficking by CK1. Overexpression of CK1 did not alter functional expression of -T663-hENaC. In contrast, modest overexpression of CK1 enhanced, while higher levels of CK1 overexpression inhibited -T663-hENaC functional expression. CK1 did not phosphorylate the C-terminus of either -T663-hENaC or -A663-hENaC in vitro. These data suggest that CK1, and perhaps specifically CK1, regulates the intracellular trafficking of the -A663T functional polymorphism of hENaC indirectly by altering the rate of -T663-hENaC biosynthesis and/or delivery to the plasma membrane.