The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction–mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca2+ waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.