© 2015 Fu et al. The ▵F508 mutant form of the cystic fibrosis transmembrane conductance regulator (▵F508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27° C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ▵F508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ▵F508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ▵F508 CFTR by ∼18% and ∼91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ▵F508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ▵F508 CFTR. To determine where and when the ubiquitination occurs during ▵F508 CFTR turnover, we monitored the ubiquitination of rescued ▵F508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ▵F508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a postendocytic compartment. This post-endocytic ubiquination of ▵F508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ▵F508 CFTR.