The 20S proteasome is part of a larger complex, the 26S proteasome, that is implicated in the ATP-dependent degradation of multiubiquitin-conjugated proteins (1). About 80% of intracellular protein breakdown occurs via the ubiquitinproteasome system (UPS). Key proteins such as transcription factors, nuclear receptors, cyclins, cyclin-dependent kinase inhibitors, p53, and NF-κB are regulated by this pathway. Thus, the UPS has been implicated to play a role in multiple cellular events including the cell cycle, signal transduction, antigen presentation, and DNA repair and transcription (2, 3). In 1984 Varshavsky and co-workers discovered that ubiquitin-dependent pathways play a role in cell cycle control, and suggested that protein degradation is instrumental in regulation of gene expression (4). Consistent with this idea, Franke and colleagues had shown that proteasomes localize to the nuclei of Xenopus laevis oocytes and HeLa cells (5, 6). Subsequent work confirmed that (i) all components of the UPS that are required for protein degradation indeed reside in the cell nucleus (7); (ii) nuclear proteins are substrates for proteasomal degradation (8); and (iii) proteasome-dependent proteolysis occurs in distinct nucleoplasmic foci (9). The intricate balance between nuclear function and quality control through proteolysis is exemplified by reports that show a correlation of aberrant nuclear protein aggregates with inhibition of transcription in neurodegenerative diseases such as Huntington's chorea and animal and cell culture models of polyglutamine repeat disorders (10,11). Considering the central role of the UPS in nuclear processes, a detailed knowledge of the time and place at which a substrate is ubiquitinylated and degraded will be essential to our understanding of the cellular mechanisms that orchestrate the expression of thousands of genes or development of subnuclear pathologies. Here, we describe fluorescence-based localization methods for proteasomes, protein aggregates, and proteasomal proteolysis in the cell nucleus that may aid to analyse the UPS in housekeeping and disease conditions. © 2008 Humana Press.