The mismatch repair (MMR) gene hMLH1 is mutated in ~50% of hereditary non-polyposis colon cancers and transcriptionally silenced in ~25% of sporadic tumours of the right colon. Cells lacking hMLH1 dis-play microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR-pro®cient and arrested at the G 2 /M cell cycle check-point following treatment with the DNA methylating agent N-methyl-N¢-nitro-N-nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR pro®ciency was restored even at low hMLH1 concentrations, while checkpoint activ-ation required a full complement of hMLH1. In the MMR-pro®cient cells, activation of the MNNG-induced G 2 /M checkpoint was accompanied by phos-phorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is function-ally inactivated in these cells by SV40 large T antigen. Keywords: cell cycle checkpoint/hMLH1/methylating agent/mismatch repair/TetOff