BACKGROUND: Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) 'prodrug' system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hypothesized that treatment of tumor cells with retinoic acid could augment the bystander erect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. METHODS: We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gone to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of honor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. RESULTS: Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin43 and was associated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no detectable connexin 43. CONCLUSION: These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.