Although the biosynthetic arrest of the DeltaF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK(1) cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane DeltaF508 CFTR to be approximately 4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.