The efficacy of HSV-TK/ganciclovir-based gene therapy on malignant gliomas largely relies on the amplitude of the bystander effect. In these experiments, the anti-inflammatory drug Sulfasalazine increased the HSV-TK/ganciclovir bystander effect in C6, 9L and LN18 cells but not in U87 glioma cells. Using bi-compartmental culture devices and conditioned medium transfer experiments, we showed that in C6, 9L and LN18 cells but not in U87 cells, Sulfasalazine also unveiled a new, contact-independent mechanism of HSV-TK/ganciclovir bystander effect. Upon treatment with ganciclovir, human LN18-TK but not U87-TK cells synthetized and released TNF-alpha in the culture medium. Sulfasalazine sensitized glioma cells to the toxic effect of TNF-alpha and enhanced its secretion in LN18-TK cells in response to GCV treatment. The caspase-8 inhibitor Z-IETD-FMK and a blocking antibody to TNF-alpha both inhibited the contact-independent bystander effect in LN18 cells. Taken together, these results suggest that TNF-alpha mediates the contact-independent bystander effect in LN18 cells. The treatment with GCV and/or Sulfasalazine of tumor xenografts consisting of a mix of 98% C6 and 2% C6-TK cells shows that Sulfasalazine is also a potent adjunct to the in vivo treatment of gliomas.