When animals are treated with carcinogenic agents that alkylate O6-guanine residues, the incidence of tumors in specific tissues often relates inversely to the level of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) present in the tissue. Similarly, the hypersensitivity to anticancer chloroethylnitrosoureas of some human tumor cell lines is believed to result from their deficiency in MGMT. We have undertaken a comprehensive investigation of MGMT expression in a panel of nine characterized human glioma cell lines. Methyltransferase activity determined by incubating protein extracts of these glioma lines with [3H]methylated DNA ranged from undetectable in six lines (the Mer- phenotype) to greater than 0.8 pmol/mg in two lines (U-373 MG and D-392 MG). MGMT protein was undetectable in Western blots of the Mer- cell extracts probed with specific anti-MGMT monoclonal antibodies. Consistent with these results, steady-state levels of MGMT mRNA, determined by Northern blot analysis, were detectable only in the three Mer+ glioma lines (U-373 MG, D-392 MG, D-263 MG). Southern analysis of EcoRI-digested DNA probed with MGMT cDNA revealed no amplification, rearrangement or deletions of the MGMT gene in any of the glioma cell lines. This is the first report that examines MGMT expression at the biochemical, molecular and genetic levels in a particular tumor type. These studies suggest that transcriptional regulation is the basis of the Mer- phenotype in these malignant human glioma cell lines, since no gross structural or quantitative abnormalities of the MGMT gene were seen in the phenotypically Mer- lines.