The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the 70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and I B, and directs the SCF β-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, togetherwith the previous SCFSkp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.