PURPOSE: 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/GCV) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin expression, we hypothesized that these compound could be used to augment the HSVtk/GCV bystander effect. MATERIALS AND METHODS: We transferred HSVtk gene to AB12 cell line that express connexin43 as a component of gap junction. We examined the effects of retinoic acid (RA) on GJIC utilizing a functional double-dye transfer study. To evaluate the bystander effect in vivo, a murine subcutaneous tumor model was established. Before proceeding with comparisons of HSVtk/GCV mediated bystander cell killing, we evaluated the effects of RA on flank tumor growth in order to rule out a potential antitumor effect of RA alone. Then we determined the effects of retinoic acid on bystander-mediated cell killing in an animal model. RESULTS: Addition of all-trans retinoic acid increased GJIC in AB12 cell line and was associated with more efficient GCV induced bystander killing in animal model. HSVtk transduced tumors in mice treated with the combination of GCV and retinoids were significantly smaller than those treated with GCV or retinoids alone. CONCLUSION: These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effects and may thus be a promising new approach to improve response in gene therapy utilizing the HSVtk/GCV system to treat tumors.