Ubiquitin serves as a molecular zipcode to direct and sort ubiquitinylated proteins into distinct biological pathways. Although novel modes of ubiquitin interaction have recently been characterized, conventional ubiquitin-binding domains (UBDs) recognize ubiquitin through a hydrophobic pocket centered around isoleucine 44 and lined by residues in beta sheets 3 and 4. In this study, we report a novel mode of interaction between ubiquitin and the cyclin-dependent kinase subunit of Saccharomyces cerevisiae, Cks1p, an adaptor protein involved in transcriptional regulation through recruitment of proteasomal subunits to gene promoters. Cks1p interacts specifically with monoubiquitin and tetraubiquitin with an affinity several orders of magnitude greater than that of other ubiquitin-binding domains and in an unconventional fashion, which differs from interactions documented so far between ubiquitin and conventional UBDs. The loop between helices alpha 1 and alpha 2, and to a minor extent the N-terminal alpha-helix of Cks1p, are involved in the interaction with the alpha-helix of ubiquitin, instead of its I44-centered hydrophobic pocket. Not only is this the first time the alpha-helix of ubiquitin is implicated in a protein/protein interaction, thereby shedding new light on the mechanisms of ubiquitin recognition, but also the first report of a direct physical interaction between ubiquitin and Cks1p, inferring a role for ubiquitin binding in the transcriptional function of Cks1p.