Background: The extracellular domain of the HER-2/neu oncogene product is increased in sera of some patients with epithelial cancers. Our aim was to develop an automated serum assay for the extracellular domain of the HER-2/neu protein. Methods: We used a monoclonal antibody labeled with fluorescein for capture and a monoclonal Fab′ fragment labeled with alkaline phosphatase for detection. Separation of bound and free detection conjugate was performed with magnetizable particles coated with monoclonal antibody to fluorescein. Alkaline phosphatase activity was measured kinetically at 405 or 450 nm. Results: The assay was linear from 0.1 to 250 μg/L. No hook effect was evident up to 10 000 μg/L. Within-run imprecision (CV) was 0.8–1.2%, and total imprecision was 1.1–1.7%. Cross-reactivity with human epidermal growth factor receptor, which has extensive homology with HER-2/neu extracellular domain, was <0.6%. Human anti-mouse antibodies, heterophilic antibodies, and rheumatoid factor did not interfere, nor did the therapeutic monoclonal antibody Herceptin®. In 51 healthy females, the mean value was 9.3 μg/L with a range of 6.4–14.0 μg/L. No reagent lot-to-lot variability was detected over four lots of reagents tested. Conclusion: The Bayer Immuno 1TM assay for HER-2/neu was precise and resistant to interferences, characteristics that are essential for longitudinal monitoring of cancer patients.