Currently, artificial binding proteins based on non-antibody scaffolds are developed successfully as therapeutic, diagnostic and analytical tools. In this work, to create artificial binding proteins several amino acid positions of a natural binding region in the beta sheet of ubiquitin were randomized. From the resulting ubiquitin library binding proteins against the pharmacologically relevant protein tumor necrosis factor (TNF)-alpha were selected by ribosome display. Detailed analysis of a variant with high affinity and specificity for TNF-alpha showed a 1:1 stoichiometry for binding to the homotrimeric target protein. In contrast, other TNF-binding proteins exhibit a 3:1 stoichiometry. The unusual binding behavior suggests that the ubiquitin-derived binding protein recognizes a new, uncharacterized epitope on TNF-alpha and underlines the potential of artificial binding proteins to target epitopes, which might by hardly accessible to antibodies.