The 20S core of the proteasome, which together with the regulatory particle, plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically the role of the antechambers is not fully understood and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we show that by applying both electron microscopy and tandem MS approaches to this multi-subunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allow us to conclude that a maximum of three green fluorescent protein (GFP) and four cytochrome c (Cyt c) substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover we deduce that one GFP or two Cyt c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases were translocation rates are slower then proteolysis. More generally the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.