ERBB2 (HER2/Neu) gene amplification and overexpression is associated with increased risk of metastases and shorter survival in breast cancer. Tyrosine 1248 is a major phosphorylation site of ERBB2 and reflects the activation status of the receptor. The aim of this study was to investigate the relationships between quantitative levels of pY1248-ERBB2 (p-ERBB2) and the expression of epidermal growth factor receptor (EGFR)-family members, and whether p-ERBB2 could provide additional prognostic value compared with established prognostic markers. For this purpose we developed a highly sensitive chemiluminescence-linked immunoassay (CLISA) and detected p-ERBB2 levels in 70 primary breast cancer biopsies. Phosphorylated ERBB2 correlated with EGFR and ERBB2, and inversely with oestrogen receptor (ER), progesterone receptor (PgR) and ERBB4 expression levels. Additionally, p-ERBB2 was associated with poor clinical outcome in univariate and multivariate Cox regression analysis. Further studies are needed to evaluate the predictive value of p-ERBB2.