The ubiquitin/proteasome pathway is a well characterized system for degrading intracellular proteins, although many aspects remain poorly understood. There is, for instance, a conspicuous lack of understanding of the site(s) where nuclear proteins are degraded because the subcellular distribution of peptidase activity has not been investigated systematically. Although nuclear proteins could be degraded by importing proteasomes into the nucleus, it is also evident that some nuclear proteins are degraded only after export to cytosolic proteasomes. Proteasomes and substrates are mobile, and consequently, the sites of degradation might not be static. We sought to identify the location of proteasomes to provide more conclusive evidence on the sites of protein degradation. We report that catalytically active proteasomes exist almost exclusively in the cytosol. The resulting lack of nuclear peptidase activity suggests that little, if any, degradation occurs in the nucleus. These and other studies suggest that the export of proteolytic substrates could define an important regulatory step in the degradation of nuclear proteins by cytosolic proteasomes.The ubiquitin/proteasome pathway is a major mechanism for eliminating regulatory and damaged proteins. The key enzymology is well understood, and many targeting factors that attach ubiquitin to proteolytic substrates have been identified and characterized (1). Despite these advances, there remain areas of ambiguity. A detailed understanding of the assembly of multiubiquitin chains, the transport of proteolytic substrates, and sites of intracellular protein turnover remain unclear.Many proteins that are involved in cell cycle progression, DNA repair, and transcription are nuclear proteins that play a central role in cell growth and stress response. The stability of these proteins is controlled by the proteasome, and it is generally assumed that they are degraded inside the nucleus. This view is fostered by the detection of proteasome subunits in the nucleus (2, 3) and enrichment in the nuclear envelope (4). However, many of these studies examined the distribution of GFPtagged proteins, which does not ensure that the tagged subunits are present in intact proteasomes. This is an important consideration because certain proteasome subunits and subcomplexes perform non-proteolytic roles in the nucleus (5, 6). We also note that certain GFP-tagged proteasome subunits are not efficiently assembled into intact complexes, and in some instances the fluorescence is reduced after assembly into the proteasome (7). Consequently, the signal observed could arise predominantly from the free form of proteasome subunits. Critically, there is no convincing evidence that peptidase activity is present in the nucleus.We and others reported that the yeast DNA repair protein Rad4, DNA polymerase subunit Cdc17 (2), and HO endonuclease (8) are stabilized in nuclear export mutants, although proteasome assembly and catalytic activity are unaffected. The stabilizing effect of blocking export is not restri...