An antibody preparation specific for human O6-methylguanine-DNA methyltransferase (EC 220.127.116.11) was obtained by immunoaffinity purification on two types of affinity columns with the purified human and mouse methyltransferase proteins as ligands. The antibodies were used in Western blotting analysis of fractionated cell extracts. More than 90% of the methyltransferase protein was recovered in the cytoplasmic fractions with both human HeLa S3 cells and MR-M cells, the latter overproducing the enzyme 36 times as much as the former. Cytoplasmic localization of the methyltransferase in HeLa S3 cells was further confirmed by in situ immunostaining. By Western blotting analysis of fractionated cell extracts from HeLa S3 cells treated with alkylating agents, we found that amounts of the enzyme decreased more rapidly in the nuclear fraction than in the cytoplasmic fraction, and recovery of the enzyme level in the cytoplasmic fraction was slower than that in the other. These results suggest that the methyltransferase protein is degraded in the nucleus after it commits the repair reaction and that the cytoplasmic enzyme is transported into the nucleus as the nuclear methyltransferase is used up in this manner.