In addition to its synthesis in the developing placenta, human CG or the isolated alpha- and beta-subunits are synthesized by a wide variety of both trophoblastic and nontrophoblastic tumor cell lines. The beta-subunit confers unique biological activity on the hormone and is encoded by a family of six genes or pseudogenes linked physically with beta LH at one locus on chromosome 19. Previous work has demonstrated that members of the beta CG family are not expressed to the same extent in first-trimester placenta. The factors allowing differential expression of the six nearly identical genes are unknown. It is also undetermined which of the multiple beta CG genes are actively expressed in the tumor cell lines that produce beta-subunit ectopically. One potential mechanism for control of beta CG gene expression is DNA methylation. A unique method of two-dimensional DNA electrophoresis was used to analyze the methylation status of each of the individual beta CG genes in placenta and in tumor cell lines that either do or do not express the protein. The DNA from each source exhibited unique, reproducible methylation patterns over the six beta CG genes. Particularly interesting were the observations that: 1) despite their nearly identical sequences and close chromosomal proximity, the six beta CG genes are distinguished from one another within the same and different cell types by their extent and pattern of methylation; 2) the beta CG locus is significantly hypomethylated in placenta, a state maintained in choriocarcinoma cells (JAr); 3) the beta CG gene cluster is unmethylated at a limited set of sites in DNA from both 2RA (a transformed fibroblast cell line that does not produce beta-subunit) and CBT (a glioblastoma cell line that produces beta-subunit ectopically), suggesting that general demethylation of the domain does not accompany activation of the locus in CBT but rather that site-specific changes may be responsible, at least in part, for inappropriate beta CG expression; and 4) the ratio of beta CG gene 3 transcripts to beta CG gene 5 transcripts is at least 10-fold higher in CBT cells than in JAr cells, suggesting that the reduced methylation in CBT cells of gene 3 compared to gene 5 may be a factor contributing to the activity of these genes.