To distinguish cystic fibrosis transmembrane conductance regulator (CFTR) at the surface of epithelial cells from that present in intracellular membranes, intact T84 cells were treated with periodate and biotin-LC- hydrazide to derivatize exposed glycoconjugates. Cell lysates were then passed over a monomeric avidin column, which allows reversible avidin-biotin binding. After washing, biotinylated molecules were eluted with 2 mM biotin. CFTR was then immunoprecipitated with a mouse monoclonal antibody from both the unbound and biotin eluent fractions, radioactively labeled by in vitro phosphorylation, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Nonimmune mouse immunoglobulin G failed to precipitate any CFTR, and CFTR was detectable only in the wash fractions when cells were periodate treated but not labeled with biotin hydrazide. In biotinylated cells, CFTR levels were approximately equal in the unbound fraction and the biotin eluent. The proportion of biotinylated CFTR did not significantly increase when cells were labeled after treatment with 10 μM forskolin. These data demonstrate that in T84 cells CFTR is constitutively expressed on the cell surface and that activation of CFTR does not primarily depend on the cAMP-dependent trafficking of CFTR to the plasma membrane. The large unbiotinylated pool of maturely glycosylated CFTR suggests that CFTR resides in an intracellular compartment as well as being present at the cell surface.