Gap junctional intercellular communication (GJIC) has been shown to be involved in the bystander effect through herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy. In this study, we examined the expression of connexins, the components of gap junction, and the degree of GJIC in esophageal cancer cell lines and compared the bystander effect in cells with different capacities of GJIC. We found loss in connexin 26 expression and reduced connexin 43 in esophageal cancer. GJIC capacity varied among cell lines and was dependent on the connexin 43 expression in the cell-cell contact areas. In mixing assay, the extent of the bystander effect was tightly correlated with the degree of GJIC capacity. The effects of retinoic acid and cAMP on the bystander effect were also investigated. Treatment with retinoic acid, but not with cAMP, was associated with augmented bystander killing by increase in GJIC in some esophageal cancer cell lines. Our results indicated that the degree of GJIC was predictive to identify a tumor as suitable for gene therapy with the HSV-tk/GCV system. Also GJIC chemically-enhanced with retinoic acid might be useful to improve response in suicide gene therapy.