SN1-type alkylating agents that produce cytotoxic O6-methyl-G (O6-meG) DNA adducts induce cell cycle arrest and apoptosis in a manner requiring the DNA mismatch repair (MMR) proteins MutSα and MutLα. Here, we show that checkpoint signaling in response to DNA methylation occurs during S phase and requires DNA replication that gives rise to O6-meG/T mispairs. DNA binding studies reveal that MutSα specifically recognizes O6-meG/T mispairs, but not O6-meG/C. In an in vitro assay, ATR-ATRIP, but not RPA, is preferentially recruited to O6-meG/T mismatches in a MutSα- and MutLα-dependent manner. Furthermore, ATR kinase is activated to phosphorylate Chk1 in the presence of O6-meG/T mispairs and MMR proteins. These results suggest that MMR proteins can act as direct sensors of methylation damage and help recruit ATR-ATRIP to sites of cytotoxic O6-meG adducts to initiate ATR checkpoint signaling.