AbstractVarieties of cell–matrix or cell–cell adhesions are associated with the actin cytoskeleton. However, for gap junctions, which are both channels and adhesions, there has been little evidence for such an association. The purpose of this study was to determine if connexin 43 (Cx43) associates with actin and to determine if this association is altered under mechanical load in tenocytes, a mechanically sensitive cell. Avian tenocytes were subjected to multiple cyclic strain regimens and then fixed and examined immunohistochemically at various times poststrain to determine where Cx43 protein was localized within the cells in relation to actin filaments. Four percent of tenocytes had colocalization of actin filaments and Cx43, which was significantly increased with 5% cyclic strain. To confirm this phenomenon, human tenocytes and COS‐7 cells were also subjected to cyclic strain and then fixed at the same times after strain. As with avian tenocytes, Cx43 was colocalized with actin in human tenocytes and COS‐7 cells. The colocalization increased significantly after cyclic strain in human tenocytes but not in COS‐7 cells. The lack of detectable changes in COS‐7 cells may indicate that they are less mechanosensitive than tenocytes perhaps due to the less robust actin cytoskeleton seen in the COS‐7 cells when compared to the tenocytes. Furthermore, inhibiting myosin II activity greatly reduced the immunohistochemically‐detectable Cx43 on actin filaments. Connexins may associate with actin to stabilize gap junctions at the plasma membrane, ensuring that tenocytes remain coupled during periods of prolonged or intense mechanical loading. Cell Motil. Cytoskeleton 2007. © 2006 Wiley‐Liss, Inc.