Amphiregulin is a recently described member of the epidermal growth factor family. Primary breast cancers were assessed for expression of amphiregulin by immunochemistry (111 cases), Northern, and/or dot blots (68 cases). Epidermal growth factor and estrogen receptors were measured in all cases. p53 and erbB-2 expression was assessed by immunohistochemistry for most cases. There was no association of these factors with amphiregulin expression, which was detected by immunochemistry in 40 of 111 cases. A significant association of amphiregulin expression assessed by Northern dot blots versus immunochemical staining was seen (P = 0.0016). Expression was not detected in adjacent nontumor tissue by immunochemistry. Amphiregulin was expressed in tumor epithelium, but not stromal or inflammatory cells. Expression was more common in lymph node positive cases (23 of 49; 47%) than lymph node negative cases (11 of 42; 26%; P = 0.04). The coexpression of epidermal growth factor receptor and amphiregulin in 35% of epidermal growth factor receptor positive cases raises the possibility of an autocrine loop in this subset of patients. Amphiregulin stimulates fibroblast growth and is up-regulated in breast cancer. A possible effect on tumor stroma may relate to the association with metastases.