In the past, we transfected C6 glioma cells with connexin43 cDNA, resulting in a significant increase in connexin43 mRNA and protein, as well as reduced proliferation and tumorigenesis. To investigate the morphological aspects of increased connexin43 expression in these cells, we have used a combination of immunocytochemistry, cytochemistry, and electron microscopy. By confocal immunofluorescence microscopy, connexin43 protein was localized to the plasma membrane of transfected cells and extensive intracellular accumulations of connexin43 were also demonstrated. Freeze fracture preparations showed large aggregates of particles typical of mature gap junction plaques in the plasma membrane of these cells. Ultrastructural immunogold labeling with anti-connexin43 serum revealed that connexin43 protein was present in gap junctions in the plasma membrane, some of which were found in proximity to clathrin-coated pits. In addition, various intracellular membranous profiles were immunoreactive for connexin43, including annular profiles, some with fuzzy coats and some associated with lysosome-like structures. Enzyme cytochemistry revealed that these annular gap junction profiles were often associated with acid phosphatase-positive lysosomes. These studies on the intracellular localization of gap junction protein in connexin43-transfected cells are consistent with the functional expression of the transfected connexin43 cDNA and provide a useful model to study the pathways of gap junction assembly and degradation.