Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids [130][131][132][133][134][135][136] ). Cx43del [130][131][132][133][134][135][136] expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del 130-136 blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del 130-136 and Cx43 colocalized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when coexpressed with Cx43del 130-136 . Cx43del 130-136 co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del 130-136 produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.