In earlier transmission electron microscopic studies, we have described pentilaminar gap junctional membrane invaginations and annular gap junction vesicles coated with short, electron-dense bristles. The similarity between these electron-dense bristles and the material surrounding clathrin-coated pits led us to suggest that the dense bristles associated with gap junction structures might be clathrin. To confirm that clathrin is indeed associated with annular gap junction vesicles and gap junction plaques, quantum dot immuno-electron microscopic techniques were used. We report here that clathrin associates with both connexin 43 (Cx43) gap junction plaques and pentilaminar gap junction vesicles. An important finding was the preferential localization of clathrin to the cytoplasmic surface of the annular or of the gap junction plaque membrane of one of the two contacting cells. This is consistent with the possibility that the direction of gap junction plaque internalization into one of two contacting cells is regulated by clathrin. (c) 2008 Elsevier Inc. All rights reserved.