Human ClC-6 is a membrane protein belonging to the ClC chloride channel family which in mammals contains both plasma membrane and intracellular channels. Based on its primary structure ClC-6 is most closely related to ClC-7, a lysosomal chloride channel in mammals. Despite being cloned for nearly a decade, the cellular function(s) and the intracellular distribution of ClC-6 remain largely unknown. The aim of this study is therefore to investigate the expression pattern, the subcellular location and function of the human ClC-6. As a first approach we raised rabbit polyclonal antibodies against a unique region in the COOH-terminus of hClC-6. To test the polyclonal antiserum by Western Blotting, COS-cells were transiently transfected with a hClC-6 expression vector and microsomal membranes were prepared. After SDS-PAGE and Western blotting, immunostaining with the polyclonal antiserum and peroxidase-conjugated secondary antibodies recognised a band of 100 kDa (predicted molecular mass of non-glycosilated hClC-6 is 97 kDa; three independent experiments). Two observations indicate that the immunoreactive band corresponds to hClC-6. First, there was no signal on blots containing microsomes from non-transfected COS-cells. Second, the 100 kDa band was not observed when blots with microsomes of hClC-6 transfected COS-cells were incubated with pre-immune serum and peroxidase-conjugated secondary antibodies. Furthermore, after PNGaseF treatment of microsomes from transfected COS cells we observed a downward shift of the 100 kDa band on Western blot, which indicates that hClC-6 is glycosylated. In a next series of experiments we explored whether affinity-purified anti-hClC-6 antibodies could be used for immunofluorescence with Alexa Fluor dye conjugated secondary antibodies. Immunostaining of COS-cells revealed a fluorescent signal in the perinuclear region of hClC-6 overexpressing COS cells, but no signal in non-transfected wild-type COS cells. In a second series of experiments we have studied the expression pattern of ClC-6 in the mouse kidney by RT-PCR experiments on microdissected fragments of the mouse nephron. This showed a high expression of mClC-6 mRNA in the proximal convoluted part of the proximal tubulus with decreasing levels of expression in the distal parts of the nephron. From these experiments, we conclude that we have generated a polyclonal antibody which recognizes hClC-6 in Western blots and immunofluorescence and that in transiently transfected COS cells hClC-6 occurs as a glycosilated protein that resides in an intracellular, perinuclear compartment.