Previous molecular cytogenetic studies in breast cancer revealed numerous chromosomal changes and identified alterations involving the chromosomes 1 and 16 as early incidents in mammary carcinogenesis. Since both chromosomes reveal pericentromeric heterochromatic areas, these chromosomal alterations might result from instable heterochromatin caused by DNA hypomethylation. In the present study, we investigated whether hyperplastic and neoplastic lesions of the breast differ regarding the distance between the heterochromatic areas of chromosomes 1 and 16 within the nuclei. We hybridized differently fluorescence-labeled DNA samples specific for the heterochromatic regions of chromosomes 1 and 16 to formalin-fixed tissue sections. Histological classification of the lesions was supported by immunohistochemical staining using cytokeratin-specific antibodies. The methylation state of the heterochromatic regions was tested by staining with an antibody specific for methylated cytidin. Our results revealed an increased frequency of paired intranuclear signals specific for chromosomes 1 and 16 in neoplastic lesions (atypical ductal hyperplasia, ductal carcinoma in situ) compared to ductal hyperplasia and normal glandular epithelium. Staining with the methylation-specific antibody reavealed a weaker staining in neoplastic lesions compared to hyperplastic lesions and normal cells. We conclude that atypic ductal hyperplasia represents the histomorphological equivalent for the beginning of tumor genome evolution that progresses in ductal carcinoma in situ and infiltrating carcinoma.