Connexin43 (Cx43) is an integral plasma membrane protein that forms gap junctions between vertebrate cells. We have used sucrose gradient fractionation and chemical cross-linking to study the first step in gap junction assembly, oiigomerization of Cx43 monomers into connexon channels. in contrast with other plasma membrane proteins, multisubunit assembly of Cx43 was specifically and completely blocked when endoplasmic reticulum (ER)-to-Golgi transport was inhibited by 15% incubation, carbonyi cyanide mthlorophenyihydrazone, or brefeidin A or in CHOceil mutants with temperature-sensitive defects in secretion. Additional experiments indicated that connexon assembly occurred intraceiiuiarly, most likely in the trans-Goigi network. These results describe a post-ER assembly pathway for integral membrane proteins and have implications for the relationship between membrane protein oligomerization and intracellular transport.