ClC-K chloride channels are essential for sodium chloride resorption in the loop of Henle of the kidney and for secretion of potassium by the stria vascularis of the inner ear. Renal and inner ear chloride channels consist of pore-forming ClC-Ka and ClC-Kb subunits and the accessory subunit barttin. Barttin improves the stability of ClC-K channel protein, stimulates the exit from the endoplasmic reticulum and insertion into the plasma membrane and changes its function. Human ClC-K channels (ClC-Ka and ClC-Kb) are only functional in presence of their accessory subunit barttin. It is still insufficiently understood how barttin exerts the variety of functions on ClC-K proteins. We performed a tryptophan scan of barttin to define the interaction site of barttin with the channel protein. Tryptophan exhibits a bulky side chain that is expected to interfere with subunit interactions if made at positions in close proximity with the binding partner. At lipid-exposed positions tryptophan has normally no functional consequences. Barttin consists of two transmembrane helices that fulfill chaperone function for channel trafficking and a short segment close to the second helix that is important for channel activation. We substituted each single amino acid of the second helix by tryptophan, co-expressed mutant barttin and V166E rClC-K1 in tsA201 cells and investigated functional properties using conventional whole cell patch clamp techniques. Rat ClC-K1 is a homolog of human ClC-Ka that is functional also in the absence of barttin. Barttin constitutively opens the slow gate of ClC-K1 channels. With most barttin mutations V166E rClC-K1 currents were comparable to currents in the presence of WT barttin. Only point mutations M40W, G41W, M44W and V45W failed to activate the slow gate at positive potentials, similar to V166E ClC-K1 currents in the absence of barttin. Confocal imaging with fluorescence tagged proteins demonstrated surface membrane insertion for each barttin mutant, but chaperone function of mutants M40W, G41W, M44W and V45W was disabled. We confirmed this dysfunction by a second approach using concatamers of V166E ClC-K1 and barttin. For the concatamer with WT barttin trafficking and channel activation was intact. Concatamers including M40W, G41W, M44W and V45W barttin, respectively, did not activate V166E ClC-K1 channels. We conclude that the amino acids at positions 40/41 and 44/45 of the second transmembrane helix of barttin interact with ClC-K channels.