Smad4 is a key tumor suppressor that is frequently deleted or inactive in pancreatic and colon tumors. In this report, we describe an approach for attaining selective killing of Smad4-deficient tumor cells. Using a vector system involving a designed repressor with zinc finger binding domains and the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene," we demonstrate Smad4-responsive regulation of HSV-TK expression and consequent altered susceptibility to the prodrug ganciclovir (GCV). In pancreatic tumor cell lines stably transfected with the vector system, a robust differential of HSV-TK expression and GCV toxicity was attained depending on the presence or absence of cotransfected Smad4. In matched colon tumor cell lines lacking Smad4 or expressing physiological levels of Smad4, an adenoviral version of the vector system attained a significant degree of preferential killing of Smad4-negative tumor cells in response to GCV. These findings demonstrate the possibility of achieving selective killing of pancreatic and colon cells depending on their Smad4 status.