The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.