The Three Rs (Reduction, Refinement, Replacement) concept of Russell & Burch in relation to humane laboratory animal experimentation is introduced, and special aspects concerning the testing of biologicals are outlined. The role of ECVAM in promoting the Three Rs in the European Union, by organising workshops and task forces, supporting conferences, and financing and/or participating in alternative test development, pre-validation and validation, is reviewed. Finally, examples are given of biologicals-related issues which deserve attention or greater focus.