The presence of human epidermal growth factor type 2 (HER2) on 20-30% of human breast cancer is a prognostic indicator of more rapid disease progression and a therapeutic indicator for anti-HER2 monoclonal antibodies. Because the literature has demonstrated some discordance between primary and metastatic tumors in the same patient for expression of the HER2 marker, we set out to develop an imaging agent that could be used to assess the marker concentration in vivo in an individual patient. The pharmaceutical company Affibody AB has optimized the specificity of Affibody molecules for HER2. Two Affibody molecules, a 7 kD and an 8 kD protein, were designed with a single carboxy terminal cysteine in order to provide a specific location for the purposes of labeling for various types of imaging. We have prepared N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide utilizing a coupling reaction between [(18)F]fluorobenzoic acid and aminoethylmaleimide. We then optimized the conjugation of this radiolabeled maleimide to the free sulfhydryl of cysteine by incubating at pH 7.4 in phosphate buffered saline containing 0.1% sodium ascorbate. An overall uncorrected yield of radiolabeled Affibody molecule of approximately 10% from [(18)F]fluoride was achieved in a 2 h synthesis. These conjugated Affibody molecules were obtained with a specific activity of 2.51 +/- 0.92 MBq/microg. Characterization of the product by HPLC-MS supported the conjugation of [(18)F]FBEM with the Affibody molecule. The radiolabeled Affibody molecule retained its binding specificity as demonstrated by successful imaging of xenografts expressing HER2.