EGFRvIII was first reported in human glioblastomas. Subsequent reports indicated EGFRvIII protein to be frequently detected in several other human cancers, but not in normal tissues. Our previous studies suggested that EGFRvIII could induce a transformation from ligand-dependent non-tumorigenic cell line to ligand-independent malignant phenotype cells in vitro and in vivo. Transfection of EGFRvIII in MCF-7 cell line resulted in a 3-fold increase in colony formation and significantly enhanced tumorigenicity in nude mice (p < 0.001). EGFRvIII could also induce ErbB-2 phosphorylation. The existence and significance of EGFRvIII transcript in human breast cancer, however, was not reported. In our study, we detected the presence of EGFRvIII mRNA and revealed a high incidence (67.8%) of EGFRvIII transcript in human primary invasive breast cancer by utilizing laser capture microdissection (LCM)/RT-PCR to capture pure breast cancer cells. In addition, 57.1% of the infiltrating breast carcinomas expressed both EGFRwt and EGFRvIII mRNA in the same tumor. There is no detectable EGFRvIII mRNA in normal breast tissue. Evaluation of the EGFRwt and EGFRvIII protein levels in the same sample sets by immunohistochemical analysis further confirmed the LCM/RT-PCR finding. Our study provides first direct evidence of high incidence of coexpression of EGFRvIII and EGFRwt in human invasive breast cancer tissue. The unique characteristics and high prevalence of EGFRvIII in invasive human breast cancer as well as negative expression in normal breast may suggest its important role in breast carcinogenesis and make it an ideally potential target for treatment of breast cancer without interrupting normal EGFR signaling.