Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. Though the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multi-subunit channel may be more physiologically relevant than the stability of individual subunit proteins, we studied the stability of ENaC by recording single channel activity in over 400 A6 cells with the translation blockers and protein trafficking inhibitors. The data suggests that the half-life of ENaC channels is approximately 3.5 hrs following puromycin, BFA and nocodazole treatment. Furthermore, these three drugs had no significant effect on the open probability of ENaC for at least 6 hrs after exposure. A decrease in apical channel number and open probability was observed following 2 hrs of cyclohexamide treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease, furin, and therefore, changes in protease activity can not explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1 hr exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.