The nuclear proteasome activator REGγ/PA28γ is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGγ-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.