Increasing evidence suggests that proteasomal dysfunction plays a role in the pathogenesis of Lewy body diseases. We have used pharmacological inhibitors of the proteasome to model proteasomal dysfunction in cultured rat cortical neurons. Proteasomal inhibition induced apoptotic death and formation of cytoplasmic ubiquitinated inclusions, which were present only in viable neurons. Actinomycin D, but not a caspase inhibitor, prevented inclusion formation, whereas both agents inhibited cell death. alpha-Synuclein and thioflavin S staining were found within the inclusions. alpha-Synuclein, however, did not appear to be ubiquitinated or aggregated. A dominant-negative mutant of an E2 ubiquitin-conjugating enzyme, cdc34, prevented inclusion formation and attenuated cell death. Our results suggest that in cortical neurons: (a) proteasomal dysfunction plays a role in formation of ubiquitin/alpha-synuclein-positive inclusions, (b) inclusion formation is an active cell process requiring transcription, and (c) ubiquitination of certain proteins is required for inclusion formation and may participate in neuronal death.