Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aβγ channels exhibited increased currents compared with αT663βγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aβγ and αT663/C664Mβγ channels. The decrease in functional expression of αT663/C664Mβγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.