Suicide gene therapy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) is notable for producing multi-log cytotoxicity in a unique pattern of delayed cytotoxicity in S-phase. As hydroxyurea, a ribonucleotide reductase inhibitor that activates mismatch repair, can increase sensitivity to GCV, we evaluated the role of MLH1, an essential mismatch repair protein, in GCV cytotoxicity. Using HCT116TK (HSV-TK-expressing) colon carcinoma cells that express or lack MLH1, cell-survival studies demonstrated greater GCV sensitivity in the MLH1-deficient cells, primarily at high concentrations. This could not be explained by differences in GCV metabolism, as the less sensitive MLH1-expresssing cells accumulated more GCV triphosphate and incorporated more of the analog into DNA. SiRNA suppression of MLH1 in U251 glioblastoma or SW480 colon carcinoma cells also enhanced sensitivity to high concentrations of GCV. Studies in a pa nel of yeast deletion mutants confirmed the results with MLH1, and further suggested a role for homologous recombination repair and several cell-cycle checkpoint proteins in GCV cytotoxicity. These data suggest that MLH1 can prevent cytotoxicity with GCV. Targeting mismatch repair-deficient tumors may increase efficacy of this suicide gene therapy approach to cancer treatment.