Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (delta F508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both delta F508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis.