ClC-2, a plasma membrane chloride channel, is widely distributed in different organs and tissues. Functionally, it has been proposed to play roles in transepithelial fluid transport and chloride homeostasis in neurones. In the intestinal epithelia and polarised cell lines, the channel is located in the basolateral membrane, presenting in addition an intracellular vesicular pattern of expression. The goals of the present study were, 1) to identify the intracellular pool in which ClC-2 is located using markers of the secretory and endocityc pathways, and 2) to evaluate the dynamic of the protein in the plasma membrane. HEK-293 cells were transiently transfected with hClC-2/EGFP (Green Fluorescent Protein, EGFP, in the C-terminus) or hClC-2/HA (Hemagglutinin peptide epitope, HA, in an extracellular loop between the L and M transmembrane domains). Both constructs behaved like the wild type channel: HEK-293 cells transiently transfected with either construct displayed typical slow currents activated by membrane hyperpolarization; and destination experiments in MDCK cells revealed the usual basolateral localisation. Confocal microscopy studies with markers of early and recycling endosomes (syntaxin-13 and transferrin receptor), late endosomes and lisosomes (lysotracker), endoplasmic reticulum (pEYFP-ER) and Golgi apparatus (pEYFP-Golgi), did not show any significant colocalization with the channel protein. All these experiments were repeated using at least three different cell cultures. To determine the dynamics of the channel protein at the plasma membrane surface, we treated the HEK-293 transfected cells with beta-cyclodextrin (βCD) and followed the localisation of the constructs by confocal microscopy or surface protein biotinylation. Using either construction, incubation with 10 mM βCD during 30 min at 37°C increased significantly the expression of channel at the cell surface. The observed increase in biotinylation experiments was 2.6±0.2-fold (mean±SD, p<0.05, 3 experiments, 2-3 replicates in each experiment). Surprisingly, in parallel patch-clamp experiments treatment with βCD decreased the current mediated by ClC-2 by 53±10% (n=5, p<0.05). Based on present results, we hypothesize that ClC-2 could be present in an intracellular ″non classical″ vesicular compartment, and that cholesterol depletion-sensitive dynamic traffic towards, and retrieval from, the cell surface accounts for the membrane ClC-2 expression in the basal state.