The lens gap junction connexin46 of rat (rCx46) is expressed in lens fibre cells and connect the cytoplasms of interacting cells. To understand the role of the C-terminus in functional expression of rCx46, different mutants were generated by truncation of the C-terminus and labelled with eGFP. After expression in HeLa cells, compartmentalization, formation of vesicles and gap junction plaques were analyzed using confocal laser scanning microscopy. Functionality of formed gap junctions channels were evaluated by Lucifer yellow dye transfer. Almost complete ablation of the C-terminus to generate the mutant rCx28.2 disrupted compartmentalization as well as formation of vesicles and functional gap junction plaques. The mutant rCx35.3 whose C-terminus is about 68 residues longer than rCx28.2 showed formation of functional gap junction plaques and compartmentalization within HeLa cells. Furthermore it was found in large vesicles which we could divide into secretory vesicles and vesicles containing annular junctions, which are used for removal of gap junctions from the plasma membrane. This mutant acted like the wild type. In contrast the mutant rCx32.6 which had a 42 residue longer C-terminus than rCx28,2 did not show such vesicle formation and compartmentalization. However large functional plaques were found. Therefore it is concluded that the C-terminus affect formation of functional gap junction channels. Moreover it is expected that both, formation and removal of rCx46 are altered.