To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-alpha (TNF-alpha), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-alpha (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-alpha was mimicked by exogenous sphingomyelinase (0.1 U/ml) and C(2)-ceramide (50 microM), but neither C(2)-dihydroceramide, a membrane-impermeable analog of C(2)-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. C(2)-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. C(2)-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GF109203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-alpha inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS.