Methylene blue in a 1% sterile solution for injection to help localize occult breast tumor was shown to interfere with the estrogen-receptor protein (ERP) binding-capacity assay. Cytosols derived from ERP-positive lyophilized powders and human breast tissue were evaluated with and without varying levels of treatment with methylene blue. Cytosols treated with 0.1% methylene blue, a clinically significant level, demonstrated a substantially lower ERP binding capacity compared with control cytosols. This alteration was found to be due to a reduction in specific binding capacity and not to an alteration in apparent cytosol protein concentration. The use of methylene blue for occult breast tumor localization is not recommended when an ERP binding-capacity assay is anticipated.