Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitin-proteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-l-leucyl-leucyl-l-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.