Gap junctions, consisting of connexins, allow the exchange of small molecules (<1 kD) between adjacent cells, thusproviding a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is amember of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearingimpairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseasesare still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cellsstably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells weretreated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or α-glycyrrhetinic acid, suggestingthat Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, α-glycyrrhetinicacid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assayrevealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstratedthat in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cellswere treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed,and possibly degraded by both proteasomal and lysosomal pathways. Gap junctions, consisting of connexins, allow the exchange of small molecules (<1 kD) between adjacent cells, thusproviding a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is a member of the connexin family. Mutations on connexin 31 However, the pathological mechanism for connexin 31 mutants in these diseases is still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cells may be more expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells weretreated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or α-glycyrrhetinic acid, suggesting that the Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin 31, as well as the formation of gap junction intercellular communication by c onnexin. Additionally, α-glycyrrhetinic acid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assayrevealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and the fluorescence staining demonstrated in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cells were treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed, and possibly degraded by both proteasomal and lysosomal pathways.