Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.