Formation of a Lys48-linked polyubiquitin chain is required for destruction of targeted proteins by the 26S proteasome, whereas formation of a Lys63-linked polyubiquitin chain is required for modulation of protein-protein interaction, enzyme activity, and intracellular localization. In addition, monoubiquitination plays key roles in endocytosis and protein trafficking. To gain a better understanding of the role of polyubiquitination, we attempted to produce monoclonal antibodies against the polyubiquitin chains, two of which were designated as FK1 and FK2 and were extensively characterized. Both FK1 and FK2 antibodies recognize the polyubiquitin moiety but not free ubiquitin, whereas FK2 antibody, but not FK1 antibody, can recognize monoubiquitinated proteins. The FK1/FK2 antibodies can be applied to ELISA for quantification of polyubiquitin chains, to immunocytochemistry for staining of intracellular polyubiquitin chains, and also to immunoaffinity chromatography for isolation of polyubiquitinated proteins. Thus, these two antibodies are useful for isolating polyubiquitin chain-tagged proteins and for probing proteins that are modified through polyubiquitination or monoubiquitination in various cells and tissues under physiological and pathological conditions.