Suicide gene therapy using the herpes simplex virus thymidine kinase (TK) gene in combination with ganciclovir (GCV) has been shown to produce therapeutic, but limited, efficacy because of poor gene transfer efficiency and reduced bystander effect. Here we report that fusion of TK to an eight-amino acid peptide from the basic domain of the human immunodeficiency virus (HIV) Tat protein significantly increases the cytotoxic efficacy of the TK/GCV system in pancreatic cancer cells. We demonstrate that Tat8-TK protein is released from the intracellular compartment of Tat8-TK-expressing cells to the extracellular medium after GCV treatment. Interestingly, we show that this conditioned medium is then able to mediate cytotoxicity of wildtype cultures, suggesting the internalization of the Tat8-TK protein. Moreover, a strong antitumoral effect of Tat8-TK/GCV treatment could be achieved by two different in vivo approaches. Tumors injected with NIH 3T3/Tat8-TK cells attached to microcarriers (MC+Tat8-TK) and treated with GCV led to a 35.6% reduction in the initial tumor volume and to 50% tumor eradication. Furthermore, electrogene transfer of TK or Tat8-TK followed by administration of high doses of GCV led to an overall statistically significant reduction in tumor growth. However, the reduction in initial tumor volume was statistically significant only for the Tat8-TK group (59.5% reduction). Moreover, in this group 50% complete tumor eradication was achieved. When moderate doses of GCV were administered, the overall reduction in tumor growth was statistically significant only in the Tat8-TK group. Therefore, our results suggest that fusion of TK to the Tat8 peptide enhances TK/GCV suicide gene therapy.