O(6)-methylguanine-DNA methyltransferase (MGMT), is a DNA repair enzyme that recognizes O(6)-alkylated guanine, a base analog resulting from treatment with alkylating agents. O(6)-6-thioguanine (6-TG) is used clinically to treat malignant as well as inflammatory diseases. Although MGMT participates in resistance to alkylating agents, it has not been shown to be involved in resistance of tumors to 6-TG. In this study we used a human melanoma cell line (GA) and its selected 6-TG drug resistant variant (GA-6-TG) to investigate whether MGMT plays a role in determining the drug resistant phenotype of GA-6-TG cells. We showed that GA-6-TG resistant cells express about three fold more MGMT protein and mRNA than GA cells. Treatment with 6-TG diminishes significantly MGMT amounts in both cell lines. Increased amounts of MGMT in resistant cells, are consistent with hypermethylation of the MGMT gene coding-region. Pretreatment of cells with the MGMT inhibitor O6 benzyl guanine, resulted in sensitization of GA-6-TG cells to 6-TG. Taken together, our data suggests that MGMT is associated with 6-TG drug resistance. In analogy to patients treated with alkylating agents, patients with tumors containing increased MGMT amounts, may be more resistant to 6-TG and therefore may benefit from treatment with MGMT inhibitors.