The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of α, precursor β and processed β subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of α and precursor β but no processed β subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is∼ 300 kDa. Radioactivity chases from these complexes into mature, long-lived∼ 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing α subunits and the other, precursor β subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor β subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the β subunits and juxtaposition of the two β rings) is accompanied by conformational changes in the (outer) α rings, and may be inefficient. Finally, interferon-γ had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.