AbstractGap junctions are intercellular channels formed by hemichannels (or connexons) from two neighboring cells. Hemichannels, which are composed of proteins called connexins, can function as conduits of ATP and glutamate, and interact with adhesion molecules and other signaling elements. As a result, their functional repertoire is expanding into other roles, such as control of cell growth or cell migration. Here we further elucidate the involvement of hemichannels in cell–cell adhesion by analyzing how connexins regulate cell adhesion without the need of gap junction formation. Using a short‐term aggregation assay with C6‐glioma and HeLa cells stably transfected with connexin (Cx) 43 or Cx32, we found that the connexin type dictates the ability of these cells to aggregate, even though these two cell types do not usually adhere to each other. We have also found that high expression of Cx43, but not Cx32 hemichannels, can drive adhesion of cells expressing low levels of Cx43. Aggregation was not dependent on high levels of extracellular Ca2+, as Ca2+ removal did not change the aggregation of Cx43‐expressing cells. Our data confirm that connexin hemichannels can establish adhesive interactions without the need for functional gap junctions, and support the concept that connexins act as adhesion molecules independently of channel formation. © 2008 Wiley‐Liss, Inc.