ErbB4 is a receptor tyrosine kinase that can signal by a mechanism involving proteolytic release of intracellular and extracellular receptor fragments. Proteolysis-dependent signaling of ErbB4 has been proposed to be enhanced in breast cancer, mainly based on immunohistochemical localization of intracellular epitopes in the nuclei. To more directly address the processing of ErbB4 in vivo, an ELISA was developed to quantify cleaved ErbB4 ectodomain from serum samples. Analysis of 238 breast cancer patients demonstrated elevated quantities of ErbB4 ectodomain in the serum (≥40 ng/mL) in 21% of the patients, as compared to 0% of 30 healthy controls (P = 0.002). Significantly, the elevated serum ectodomain concentration did not correlate with the presence of nuclear ErbB4 immunoreactivity in matched breast cancer tissue samples. However, elevated serum ectodomain concentration was associated with the premenopausal status at diagnosis (P = 0.04), and estradiol enhanced ErbB4 cleavage in vitro. A 3.4 Å X-ray crystal structure of a complex of ErbB4 ectodomain and the Fab fragment of anti-ErbB4 mAb 1479 localized the binding site of mAb 1479 on ErbB4 to a region on subdomain IV encompassing the residues necessary for ErbB4 cleavage. mAb 1479 also significantly blocked ErbB4 cleavage in breast cancer cell xenografts in vivo, and the inhibition of cleavage was associated with suppression of xenograft growth. These data indicate that ErbB4 processing is enhanced in breast cancer tissue in vivo, and that ErbB4 cleavage can be stimulated by estradiol and targeted with mAb 1479.