Previous reports have suggested that Cx26 exhibits unique intracellular transport pathways en route to the cell surface compared with other members of the connexin family. To directly examine and compare nascent and steady-state delivery of Cx43 and Cx26 to the plasma membrane and gap junction biogenesis we expressed fluorescent-protein-tagged Cx43 and Cx26 in BICR-M1Rk and NRK cells. Static and time-lapse imaging revealed that both connexins were routed through the Golgi apparatus prior to being transported to the cell surface, a process inhibited in the presence of brefeldin A (BFA) or the expression of a dominant-negative form of Sar1 GTPase. During recovery from BFA, time-lapse imaging of nascent connexin Golgi-to-plasma membrane delivery revealed many dynamic post-Golgi carriers (PGCs) originating from the distal side of the Golgi apparatus consisting of heterogeneous vesicles and long, tubular-like extensions. Vesicles and tubular extensions were also observed in HBL-100 cells expressing a human, disease-linked, Golgi-localized Cx26 mutant, D66H-GFP. A diffuse cell surface rim of fluorescent-protein-tagged wild-type connexins was observed prior to the appearance of punctate gap junctions, which suggests that random fusion of PGCs occurred with the plasma membrane followed by lateral diffusion of connexins into clusters. Fluorescence recovery after photobleaching studies revealed that Cx26-YFP was more mobile within gap junction plaques compared with Cx43-GFP. Intriguingly, Cx43-GFP delivery and gap junction regeneration was inhibited by BFA and nocodazole, whereas Cx26-GFP delivery was prevented by BFA but not nocodazole. Collectively, these studies suggest that during gap junction biogenesis two phylogenetically distinct members of the connexin family, Cx43 and Cx26, share common secretory pathways, types of transport intermediates and turnover dynamics but differ in their microtubule-dependence and mobility within the plasma membrane, which might reflect differences in binding to protein scaffolds.