Tumor cells transduced with the herpes simplex virus thymidine kinase (HSVtk) gene are sensitive to the anti-viral drug ganciclovir (GCV). However, nearby untransduced tumor cells are also efficiently killed. The mechanism of this 'bystander effect' was studied by comparing pairs of tumor cell lines transfected with connexin genes that differed only in their degree of gap junctional communication. More efficient cell killing was uniformly seen in connexin transfectants compared with the less coupled cell lines. These results provide direct evidence that gap junctional communication plays an important role in mediating the bystander effect of the HSVtk/GCV system in vitro and have important prognostic and therapeutic implications for future gene therapy trials.