Transfer of suicide genes into tumor cells renders them sensitive to cytotoxic effects of specific prodrugs. We show here that both the herpes simplex virus thymidine kinase/ganciclovir (tk/GCV) and thymidine phophorylase/5'-deoxy-5-fluorouridine (tp/DFUR) suicide gene systems can induce cell death in tumor cells. Additionally in mixed cultures of cells with and without the suicide gene, death occurred in both cell types, indicative of a bystander effect. We demonstrate, in human and rodent cell lines, that the tk/GCV bystander effect requires gap junctional intercellular communication (GJIC). Where cultures lack GJIC, no bystander effect was observed. In communicating cultures, no correlation between level of GJIC and bystander effect was seen and this was due to high levels of tk activity. Additionally, we demonstrate that transfer of toxic metabolites from tk+ to tk- cells occurs within 2 hr of GCV application and, as no apoptosis could be detected until after this time, apoptosis is the result, not the cause, of the tk/GCV bystander effect. In the tp/DFUR system, a medium-mediated bystander effect, independent of GJIC and apoptosis, was observed. We demonstrated that combining tk/GCV and tp/DFUR suicide gene systems in culture was more effective than either therapy alone.