Abstract The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus β-TrCP) functions as the ubiquitin ligase for IκBα. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IκBα. The structural requirements for the interactions of FWD1 with IκBα and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IκBα abolished the binding of IκBα to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IκBα. The IκBα mutant D31E still exhibited binding to FWD1 and underwent ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser32 and Ser36, an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IκBα. Deletion analysis of Skp1 revealed that residues 61–143 of this protein are required for binding to FWD1. On the other hand, the highly conserved residues Pro149, Ile160, and Leu164 in the F-box domain of FWD1 were dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IκBα, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.