The HER-2heu proto-oncogene encodes a putative growth factor receptor which was shown to be amplified in 25-30% of primary human breast cancers. Amplification of the gene was associated with disease behavior and was more predictive of outcome than any other known prognostic factor with the exception of positive lymph nodes in patients with Stage 11 disease. Since this initial report, a number of studies have evaluated both amphka-tion rate and association between HER-2heu and prognosis in breast cancer with considerable variation in published results. Some of this variability may be due to the small numbers of specimens studied or to significant differences in the methods used to evaluate the gene and its product. The current study addresses both biological and clinical aspects of the gene and its alteration in human breast cancer. Sequence analysis of several c-DNA clones from actual tumors indicate that, unlike the rat mu gene, mutations in the transmembrane domain may not be an absolute requirement for alteration of the gene. Instead, the data are consistent with an alteration involving overexpression of a normal product since gene amplification is virtually always associated with high levels of expression in the malignant cells of these tumors. The gene product has a half life of 12 h and can be readily identified in both tissue homogenates and tissue sections from breast tumors. To circumvent potential statistical problems introduced by small numbers reported in earlier studies, an analysis of HER-2heu amplification was done in 668 primary tumors, 526 of which had long-term follow-up. The overall amplification rate in breast cancer as well as the association between gene amplification and disease outcome previously reported for Stage II patients was con-lirmed in this larger series. A comprehensive analysis of the gene and its products (DNA, RNA, and protein) was simultaneously performed on 187 tumors for which sufficient tissue was available. This analysis identSed several potential shortcomings of the various methods used to evaluate HER-2ineu in this disease (ie, Southern, Northern, and Western blots, and immunohistochemistry). The Western blot was most prone to errors in assessing HER-2/neu, while immunohistochemical analysis of frozen tissue was the most reliable method. This result has diagnostic implications. The data presented in this study further support the concept that the HER-2/neu gene may play a role in the pathogenesis of some human breast cancers.