We determined HER2 protein overexpression by immunohistochemical analysis and HER2 gene amplification by fluorescence in situ hybridization (FISH) in 215 formalin-fixed, paraffin-embedded breast tumors. Pathologist concordance for immunohistochemical scoring, and HER2 status concordance, as determined by immunohistochemistry and FISH, were high for immunohistochemical 3+, 1+, and 0 tumors but poor for 2+ tumors. Consensus immunohistochemical scores correlated with absolute and chromosome 17 (CEPI 7)-corrected HER2 gene copy number Among HER2-nonamplified tumors, the immunohistochemical score and mean absolute chromosome 17 (CEP17) copy number were weakly correlated. Seventeen tumors were HER2-amplified using absolute HER2 gene criteria but nonamplified when corrected for chromosome 17 polysomy (8 of these were immunohistochemical 2+). Assessment of benign epithelium within the immunohistochemical slides revealed either no staining or basolateral membrane staining, suggesting normal HER2 protein expression. Twenty tumors showing similar basolateral HER2 immunostaining were all low-moderate grade, tubule-forming, and HER2-nonamplified (17) or borderline amplified (3). Additional studies relating changes in HER2 gene content due to amplification or chromosome 17 polysomy and HER2 protein expression may be helpful to pathologists who interpret HER2 immnuohistochemical slides. Breast tumors scored at 2+ should be analyzed by FISH, preferably using a dual-probe FISH assay capable of distinguishing HER2 gene amplification from chromosome 17 polysomy.