Loss of heterozygosity (LOH) allows the expression of recessive mutation in tumor suppressor genes (TSG). Therefore, on the basis of Knudson's ‘two-hit’hypothesis for TSG inactivation, the detection of a high LOH frequency in a chromosomal region is considered critical for TSG localization. One of these LOH regions in breast cancer is 16q22. 1, which has been suggested to reflect the involvement of E-cadherin (E-cad), a cell–cell adhesion molecule. To confirm the tumorigenic role of E-cad, 81 sporadic invasive ductal carcinomas (IDCs) of the breast were tested for the ‘two hits’ required to inactivate this gene. A high frequency (37.3%) of LOH was detected in 67 informative tumors, but no mutation was found. To examine the possibility that transcriptional mechanisms serve as the second hit in tumors with LOH, specific pathways, including genetic variant and hypermethylation at the promoter region and abnormal expression of positive (WT1) and negative (Snail) transcription factors, were identified. Of these, promoter hypermethylation and increased expression of Snail were found to be common (> 35%), and to be strongly associated with reduced/negative E-cad expression (P< 0.05). However, unexpectedly, a significantly negative association was found between the existence of LOH and promoter hypermethylation (P< 0.05), which contradicts the ‘two-hit’model. Instead, since they coexisted in a high frequency of tumors, hypermethylation may work in concert with increased Snail to inactivate E-cad expression. Given that E-cad is involved in diverse mechanisms, loss of which is beneficial for tumors to invade but may also trigger apoptosis, this study suggests that maintaining a reversible mechanism, either by controlling the gene at the transcriptional level or by retaining an intact allele subsequent to LOH, might be important for E-cad in IDC and may also be common in TSGs possessing diverse functions. These findings provide clues to explain why certain TSGs identified by LOH cannot fulfil the two-hit hypothesis.