Proteasome, a high molecular weight multicatalytic protease complex,is responsible for most non-lysosomal intracellular protein degradations. The proteasome is composed of a 20 S catalytic core (20 S proteasome) and additional subunits, that are thought to be involved in the recognition of proteins or in the regulation of the protease activity of the proteasome. A 180 kD activator, named PA28 or Reg, associates with the 20 S proteasome and enhance the peptidase activity of the 20 S core enzyme. In this report, the biochemical isolation of the PA28-associated proteasome subset from the 20 S proteasome core is described, based on gradient anion exchange chromatography. The PA28-20 S proteasome subset, isolated from EBV-transformed B cells, was found to be highly enriched in the LMP2 (low molecular weight protein) subset, whereas no LMP2 was detected by immunoblotting in the PA28-20 S proteasome subset. The close correlation of expression of PA28 and LMP2, two interferon (IFN)-gamma inducible proteasome components,on a single proteasome subset suggests that PA28 may associate preferentially with LMP2-containing proteasomes, and/or this subset may have a specific role in the processing of environmental antigens.