Id (inhibitor of DNA binding) 4 is a member of the Id family of proteins (Id1-Id4), which function as dominant-negative regulators of basic helix-loop-helix transcription factors. Id factors are involved in numerous cell processes, including cell proliferation, differentiation, and tumorigenesis. We assessed the expression of Id4 messenger RNA (mRNA) in invasive mammary carcinoma from 31 patients, as well as in 21 cases of ductal carcinoma in situ, in 9 lymph node metastases, and in the morphologically normal epithelium adjacent to the carcinoma from the same subjects. In addition, we evaluated Id4 mRNA in atypical ductal hyperplasia from 5 other women and in normal breast tissue from yet another 5 women with no history of breast malignancy or atypia. The distribution of Id4 signal was assessed in relation to that of estrogen receptor (ER) in all samples and correlated with the Her-2 status of the carcinomas. Id4 mRNA was present in the normal ER-negative mammary epithelium in all cases; in contrast, the ER-positive cells present in the normal breast were Id4 negative. Id4 mRNA was not detected in atypical ductal hyperplasia, in 22 of the 23 cases of ductal carcinoma in situ, and in 27 of the 31 invasive carcinomas (P = .0008), all of which were ER positive. Conversely, 3 of the 31 invasive carcinomas were Id4 positive and ER negative. Only 1 ER-positive invasive carcinoma showed focal reactivity for Id4. The expression of Id4 in metastatic carcinoma paralleled that of the primary tumor. No correlation was apparent between Id4 and Her-2. Our data show that Id4 is constitutively expressed in the normal human mammary epithelium but is suppressed in ER-positive breast carcinomas and preneoplastic lesions. In contrast, ER-negative carcinomas appear to be Id4 positive. These results support a possible role of Id4 as a tumor suppressor factor in the human breast and suggest that the expression of Id4 in the mammary ductal epithelium may be regulated by estrogen. Further investigations are required to define the functions of Id4 in the human normal breast and in mammary neoplasia.