To determine the C-terminus regions of the rat lens fiber connexin46 (rCx46) that are involved in expression, sorting and internalization of the protein, different C-terminal truncated mutants were generated and labeled with EGFP. After expression in HeLa cells, functional formation of the gap junctions was studied by dye transfer, while the sorting and the internalization behavior were followed by cell imaging. The rCx46_28.2 with an almost completely ablated C-terminus did not form functional gap junction channels and was found neither in large vesicles nor in defined cellular compartments. The rCx46_32.6 formed functional gap junctions, but was not found in large vesicles and its compartmentalization seemed altered. The rCx46_35.3, rCx46_37.7 and rCx46_wt formed functional gap junctions, were compartmentalized and were detected in large vesicles. Live cell imaging experiments allowed an estimation of the vesicle budding rate from gap junction plaques formed by the different variants. It was found that relative to rCx46_32.6, the vesicle budding rate was increased by 5.78, 7.04, and 8.93 for rCx46_35.3, rCx46_37.7, and rCx46_wt, respectively. Additionally, in cells pairs expressing rCx46_32.6, only 2.91 annular junctions per cell pair were detected, while 6.70 and 12.95 annular junctions per cell pair were found in cell pairs expressing rCx46_35.3 and rCx46_wt, respectively. The results suggest that the first 82 residues of the C-terminus are necessary for a correct compartmentalization, sorting in large vesicles, and annular junction dependent removal of rCx46 connexins, while the first 53 residues of the C-terminus are enough for formation of functional gap junction channels.