Recent clinical data have suggested that the efficacy of tamoxifen in reducing the risk of local recurrence following lumpectomy and radiation therapy in patients with ductal carcinoma in situ (DCIS) is limited to patients with estrogen receptor (ER)-positive lesions. However, it is currently not known if HER2 protein overexpression might be associated with reduced tamoxifen benefit in patients with ER-positive DCIS, as has been suggested in patients with ER-positive invasive breast cancer and in preclinical models. Moreover, the frequency of HER2 overexpression in ER-positive ductal carcinoma in situ has not been previously evaluated in detail. To address this issue, we studied ER expression and HER2 overexpression in 148 cases of DCIS using a sensitive double immunostaining technique and assessed the frequency of ER expression and HER2 overexpression in relation to each other and in relation to DCIS grade. Overall, ER expression was seen in 114 cases (77%) and HER2 protein overexpression was seen in 42 cases (28%). Of 114 ER-positive ductal carcinoma in situ, 14 (12%) showed concurrent HER2 protein overexpression, and all 14 of these DCIS lesions were of high nuclear grade. In addition, in all 14 ER-positive DCIS cases that showed HER2 overexpression, double immunostaining demonstrated that ER and HER2 protein were coexpressed by the same neoplastic cells. We conclude that a subset of ER-positive DCIS show concomitant overexpression of HER2 protein. Whether or not HER2 overexpression is associated with a diminished response to tamoxifen in patients with ER-positive DCIS will require investigation in clinical outcome studies.