The MAGE-1 gene, expressed in some tumors of different histological origins, codes for a tumor antigen recognized by cytotoxic T lymphocytes. The gene is not expressed in normal tissues with the exception of testes. The present study was designed to investigate the relationship between methylation of the MAGE-1 promoter and inactivation of the MAGE-1 gene. We examined the extent to which MAGE-1 B'B promoter sequences are methylated in tumor-cell lines, in order to determine whether methylation correlates with MAGE-1 expression. Using methylation-sensitive restriction analysis followed by polymerase chain reaction (PCR), we found an inverse correlation between methylation of the MAGE-1 B'B region and MAGE-1 expression. An unmethylated state was identified in DNA from sperm and some tumor-cell lines of different origins. In contrast, a hypermethylation state was found in leukocytes and other MAGE-1 non-expressing cells. Furthermore, treatment with 5-aza-2'-deoxycytidine, a demethylating agent, induced MAGE-1 expression in tumor-cell lines in which we found no direct relation between transcriptional activity of the B'B region and MAGE-1 expression. Binding of the nuclear factors to the B'-methylated probe was strongly inhibited, indicating that methylation of cytosine interferes directly in the binding of transcriptional factors.