CFTR is a membrane glycoprotein which functions as an anion channel in exocrine epithelia and is mutated in the disease cystic fibrosis (CF). The most common disease causing mutation, deletion of a phenylalanine at position 508 (ΔF508), causes partial misfolding and retention of CFTR in the endoplasmic reticulum. This misprocessing reduces the amount of complex glycosylated CFTR ( “band C”) detected in Western blots, and glycosylation is often used to assay maturation of the protein after exposure to low temperature (eg 29 ^oC) or chemical chaperones. However core-glycosylated CFTR may also reach the plasma membrane after rescue and contribute to anion conductance. Recent attempts to demonstrate this trafficking have relied on conventional cell surface biotinylation by sulfo-NHS-SS-biotin. We examined this surface biotinylation method and found that core-glycosylated ΔF508-CFTR was labelled and pulled down on streptavidin beads, as were several intracellular proteins including the R domain of CFTR expressed in the cytosol as a soluble polypeptide. The R domain was detected in <1 % of cellular protein, similar to the fraction of cells that were permeable to ethidium homodimer-1 in live/dead cell assays, therefore the cytoplasmic aspect of CFTR is probably labelled by sulfo-NHS-SS-biotin in a subpopulation of leaky cells that are undergoing normal cell turnover. To study the trafficking of small quantities of immature protein to the cell surface more definitively, we inserted a target sequence for enzymatic biotinylation in the fourth extracellular loop of CFTR and ΔF508-CFTR. These constructs were specifically labelled on the cell surface and protected intracellularly within the lumen of the secretory pathway. Low temperature and chemical chaperones restored some channel activity but yielded little band C; most enzymatically-biotinylated ΔF508-CFTR that reached the plasma membrane was the immature “band B” glycoform. We then used live cell imaging to identify possible routes of CFTR trafficking. Secreted proteins usually move from exit sites in the endoplasmic reticulum (ER) to the cis-Golgi via tubulovesicular structures which comprise the ER-Golgi intermediate compartment (ERGIC), however total internal reflection fluorescence (TIRF) microscopy revealed ER-localized CFTR surprisingly close to the plasma membrane (<200 nm). The lectin ERGIC-53, a cargo carrier for glycosylated proteins, was used as an ERGIC marker. Two colour TIRF imaging revealed a close association between CFTR-mCherry and ERGIC-53-EGFP in mobile vesicles that were transported towards the cell periphery. These results suggest a mechanism for trafficking CFTR channels to the plasma membrane which bypasses the Golgi apparatus and may explain delivery of immature, core-glycosylated CFTR to the cell surface.