Gap junctional intercellular communication has numerous functions, each of which meets the particular needs of organs, tissues, or groups of cells. Connexins (CXs) are homologous four-transmembrane-domain proteins that are the major components of gap junctions. CX30.2/CX31.3 (GJC3) is a relatively new member of the CX protein family. Until now, however, the functional characteristics of CX30.2/CX31.3 have been unclear. To elucidate the properties of CX30.2/CX31.3 channels, their subcellular localization in HeLa cells, their effectiveness in dye transfer, and function on channels were investigated. In the immunofluorescent assay, cells that express CX30.2/CX31.3-GFP exhibited continuous fluorescence along the apposed cell membranes, rather than punctated fluorescence in contacting membranes between two cells. Surprisingly, dyes that can be capable of being permeated by CX26 GJ, according to a scrape loading dye transfer assay in previous studies, are impermeated by CX30.2/CX31.3 GJ, suggesting a difference between the characteristics of CX30.2/CX31.3 GJ and CX26 GJ. Furthermore, a significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration, suggesting a hemichannel-based function for CX30.2/CX31.3. Based on these findings, we suggest that CX30.2/CX31.3 shares functional properties with pannexin (hemi) channels rather than gap junction channels of other CXs.