The maturation of the eukaryotic 20 S proteasome complex occurs via 13 S and 16 S precursor complexes in a multistep assembly pathway. These precursor complexes contain alpha-subunits as well as unprocessed beta-subunit proproteins. We have purified and characterized the different proteasome assembly intermediates and analysed their ability to support beta-subunit proprotein processing in vitro. Our data show that 13 S and 16 S proteasome precursor complexes differ not only in size but also in their protein content and behaviour during hydrophobic chromatography. By establishing conditions which allowed us to analyse beta-prosubunit maturation in vitro we demonstrate that the processing of the homologous proproteins of the beta-subunits LMP2 and delta essentially takes place in 16 S precursor complexes. No proprotein processing activity was observed in 13 S precursor complexes. Furthermore, proprotein processing in vitro can be inhibited with a proteasome specific inhibitor, but with different efficiency for LMP2 and delta. A peptide, which represents the sequence of the proprotein processing site HGTT, exhibited no inhibitory effect on the processing of either subunit. These data provide further evidence that proprotein processing occurs via an autocatalytic mechanism. Our experiments also demonstrate that the chaperone protein hsc73 is associated with 16 S but not with 13 S precursor complexes. In support of the specificity of this interaction incubation with ATP leads to the dissociation of hsc73 from 16 S complexes and to the formation of high molecular weight aggregates. Prosubunit processing in isolated 16 S complexes does not, however, result in the formation of proteolytically active 20 S proteasomes which may be due to the fact that not all beta-subunits can be efficiently processed in vitro. In contrast to previous assumptions subunit processing and formation of proteolytic activity do not coincide and final 20 S complex assembly seems to represent in part a separate event which requires additional factors or proteins which are not present or active in the purified 16 S precursor complexes.