[Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). Hemichannel opening can be measured by electrophysiology, by quantifying the release of a hemichannel-permeable molecule into the extracellular medium or by quantifying the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule. As the extent of uptake of a molecule is proportional to its concentration, exposure time, temperature (these parameters are controlled) and, importantly, to the number of active hemichannels on the cell surface, uptake assays are routinely used to assess hemichannel opening. This protocol for the uptake of the fluorescent dye ethidium bromide was used with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types.