Aromatase inhibitors, such as anastrozole, are established in the treatment of hormone-dependent breast cancer. However, approximately 20% of patients treated with anastrozole do not respond, and it remains impossible to accurately predict sensitivity. Thus, novel markers to predict response are required. The K303R estrogen receptor (ER)α mutation confers resistance to tamoxifen treatment. Moreover, K303R-expressing MCF-7 cells, transfected with an aromatase expression vector and stimulated with androstenedione (an aromatase substrate), were found to be resistant to the inhibitory effect of anastrozole. The aim of this study was to verify whether the presence of the K303R ERα mutation is associated with response to 3-month neoadjuvant treatment with anastrozole (Arimidex) in a cohort of post-menopausal breast cancer patients. Of 37 patients with ER(+) tumors, 19 showed a clinical response to anastrozole and 18 were resistant. Biopsies were obtained from tumors responding to the therapy or from non-responding tumors. None carried the K303R ERα mutation. To our knowledge, this is the first study to search for K303R ERα mutations in tumors clinically responsive or resistant to an aromatase inhibitor. Lack of the mutation leads us to believe that this mutation has in vivo biological significance in only a subset of breast cancers.