Affibody molecules are a new class of small phage-display selected proteins using a scaffold domain of the bacterial receptor protein A. They can be selected for specific binding to a large variety of protein targets. An affibody molecule binding with high affinity to a tumor antigen HER2 was recently developed for radionuclide diagnostics and therapy in vivo. The use of the positron-emitting nuclide (76)Br (T(1/2) = 16.2 h) could improve the sensitivity of detection of HER2-expressing tumors. A site-specific radiobromination of a cysteine-containing variant of the anti-HER2 affibody, (Z(HER2:4))(2)-Cys, using ((4-hydroxyphenyl)ethyl)maleimide (HPEM), was evaluated in this study. It was found that HPEM can be radiobrominated with an efficiency of 83 +/- 0.4% and thereafter coupled to freshly reduced affibody with a yield of 65.3 +/- 3.9%. A "one-pot" labeling enabled the radiochemical purity of the conjugate to exceed 97%. The label was stable against challenge with large excess of nonlabeled bromide and in a high molar strength solution. In vitro cell tests demonstrated that radiobrominated affibody binds specifically to the HER2-expressing cell-line, SK-OV-3. Biodistribution studies in nude mice bearing SK-OV-3 xenografts have shown tumor accumulation of 4.8 +/- 2.2% IA/g and good tumor-to-normal tissue ratios.