Fbs1 is a recently discovered F-box protein that was proposed to recognize high-mannose-type asparagine-linked glycoprotein sugar chains. To reveal the specificity of Fbs1, Manalpha1-->6Manbeta1-->4GlcNAc(2), Manalpha1-->3Manbeta1-->4GlcNAc(2), and Manalpha1-->3(Manalpha1--> 6)Manbeta1-->4GlcNAc(2) were synthesized and their affinities for Fbs1 were evaluated in comparison with previously synthesized Man(9)GlcNAc(2) and Man(8)GlcNAc(2). These analyses revealed that Man(3)GlcNAc(2) had the strongest affinity and the chitobiose and alpha1-->6 linked Man residue are necessary for Fbs1 to recognize a sugar.