Human epidermal growth factor receptor 2 (HER2) expression is amplified in about 20% of breast cancer tumors, and evaluation of HER2 status should influence therapy selection. A critical gap in our knowledge is the real-world implementation of HER2 testing and its impact on treatment decisions for women diagnosed with breast cancer. To assess use of HER2 testing, to describe characteristics of patients who do or do not receive HER2 testing, to describe which HER2 tests were used (fluorescence in situ hybridization or immunohistochemistry), and to evaluate trastuzumab use as a function of HER2 results. The population included 6460 women diagnosed with invasive breast cancer between 1999 and 2007 at 8 geographically distributed Cancer Research Network healthcare delivery systems in the United States. Electronic records were used to identify patient and tumor characteristics and treatment with trastuzumab. Chart abstraction was performed for 400 women (50 per site) to identify receipt of HER2 testing and results. More than 90% of study participants received HER2 testing. Everyone who received trastuzumab had a HER2 test, and nearly all (>95%) who received trastuzumab had a positive HER2 test result recorded in their medical chart. Most (77%) eligible patients with a positive HER2 test result diagnosed after 2005 received trastuzumab. This study expands upon previous work in individual health plans. HER2 status has been successfully incorporated into medical practice to guide treatment decisions for breast cancer patients in diverse integrated healthcare delivery settings.