The presence of estrogen receptors (ERs), as detected by immunohistochemistry (IHC), is a weak prognostic marker of clinical outcome in breast cancer, but a strong predictive marker for response, for example, to tamoxifen-based therapy. As with all IHC markers, factors such as tissue fixation (both type and duration), the choice of antibody, and the threshold for interpretation of positive immunostaining can dramatically affect test accuracy and reproducibility. For example, optimal fixation for detection of ER requires at least 6-8 h in formalin, and the use of newer antibodies such as SP1 may identify additional patients who might benefit from hormonal therapy. Although the threshold for positivity may be as few as 1% of tumor cells showing nuclear signal, recent studies appear to demonstrate a dichotomization of ER IHC, with the vast majority of cases showing all positive or all negative results. This may be helpful in dictating the appropriateness of hormonal therapy, but quantification of ER by IHC, or other methods, may play a more important role in the future. Breast cancers with human epidermal receptor protein-2 (c-erbB-2; HER2) alterations are critical to identify because such tumors require unique treatment, including the use of targeted therapies such as trastuzumab. HER2 alterations at the DNA (amplification) and protein (overexpression) level usually occur in concert, and both fluorescence in situ hybridization (FISH) or IHC can be accurate methods to assess these alterations. However, recent studies have suggested that serious reproducibility issues exist in both FISH and IHC HER2 studies. To address this, a joint committee of both the American Society for Clinical Oncologists and the College of American Pathologists has promulgated new guidelines for HER2 testing. These include the following: (a) recommendations for tissue fixation for more than 6 and less than 48 h; (b) new scoring criteria, including a new threshold of 30% strong immunostaining for classification of 3+; (c) introduction of the term 'equivocal' to characterize HER2 studies that are 2+ by IHC and/or show HER2/chromosome 17 ratios of between 1.8 and 2.2 by FISH; (d) requirements for laboratories to validate HER2 assays, generally through the cross-testing of cases with another HER2 methodology, with laboratories required to attain 95% concordance for both positive and negative tests; (e) participation in HER2 proficiency testing.