Accurate assessment of human epidermal growth factor receptor 2 is critical for the management of patients with breast cancer. We set out to study the impact of the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines on the interpretation of human epidermal growth factor receptor 2 IHC results and its correlation with fluorescence in situ hybridization results. Invasive breast carcinomas with IHC HercepTest 3+ were retrieved from the archive of Mayo Clinic Rochester. The human epidermal growth factor receptor 2 slides were rereviewed, and results were recorded as percentage of invasive tumor cells with 3+, 2+, 1+, and 0 staining intensity. Human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization was performed on all tumors with 3+ staining in 70% or less of tumor cells. Of the 141 cases studied, 12 cases showed intense membrane staining in 11% to 30% of the invasive tumor cells and would have been scored as 2+ according to the new American Society of Clinical Oncology/College of American Pathologists guidelines. Of these 12 cases, 6 were positive for human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization (ratio >2.2), 4 cases were negative (HER2/CEP17 ratio of < 1.8), and 2 cases were equivocal (ratio of 1.8-2.2). One human epidermal growth factor receptor 2-positive case showed dramatic intratumoral heterogeneity with high-level amplification (ratio of 12.2) in the IHC 3+ area and no amplification (ratio of 1.0) in the IHC 1+/2+ areas. The 2007 American Society of Clinical Oncology/College of American Pathologists guidelines down-scored 2.8% of tumors from human epidermal growth factor receptor 2-positive (IHC 3+) to human epidermal growth factor receptor 2-negative (IHC 2+ equivocal and fluorescence in situ hybridization negative) in this study. Clinical studies are needed to determine whether the updated guidelines are better at predicting response to anti-human epidermal growth factor receptor 2 therapy.