Gap junctions, composed of Cxs (connexins), allow direct intercellular communication. Gap junctions are often lost during the development of malignancy, although the processes behind this are not fully understood. Cx43 is a widely expressed Cx with a long cytoplasmic C-terminal tail that contains several potential protein-interaction domains. Previously, in a model of cervical carcinogenesis, we showed that the loss of gap junctional communication correlated with relocalization of Cx43 to the cytoplasm late in tumorigenesis. In the present study, we demonstrate a similar pattern of altered expression for the hDlg (human discs large) MAGUK (membrane-associated guanylate kinase) family tumour suppressor protein in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg. hDlg is not required for Cx43 internalization in W12GPXY cells. Instead, hDlg appears to have a role in maintaining a cytoplasmic pool of Cx43. These results demonstrate that hDlg is a physiologically relevant regulator of Cx43 in transformed epithelial cells.