Molecular changes in the progressive state of tumorigenesis often include altered patterns of DNA methylation. Utilizing a series of breast epithelial cell lines, the overall 5-methylcytosine content in genomic DNA demonstrated an overall decrease when comparing two malignant cell lines (MCF-7 and T47D) with a mortal cell line (MCF 1 2M) and several derivative cell lines of the immortalized MCF10 cultures (MCF10A,-2A, -5A, A1neoT2, and 139B6). Further investigation on the methylation status of these cells lines indicated no difference in DNA methyltransferase activity, both at a protein and mRNA levels, in the nontumorigenic cell lines examined while activity was 3-10 fold higher in the tumorigenic lines (MCF7, T47D, SkBr3, MB-MDA-231, -468). Examination of the CpG island in the 5' promoter region of the estrogen receptor gene indicates that this region is unmethylated in the mortal and immortal nontumorigenic lines as well as the tumorigenic lines examined, with the exception of the estrogen receptor negative breast cell line MB-MDA-468 which appears to be partially methylated at this site. These results indicate methylation of this CpG island does not account for the inactivation of the estrogen receptor gene in immortalized nontumorigenic breast cells, suggesting another mechanism of transcriptional inactivation of ER in this environment.