The 20S proteasome of eukaryotes is an abundant multicatalytic/multifunctional proteinase complex composed of an array of nonidentical subunits which are encoded by alpha- or beta-type members of the proteasomal gene family. In budding yeast, 14 subunits had been detected and 12 proteasomal genes had been cloned and sequenced so far. Starting from peptide sequences of purified subunits of the yeast 20S proteasome, we cloned two additional proteasomal genes, PRE5 and PRE6, which both encode essential alpha-type subunits. Sequence comparison of all known eukaryotic proteasomal proteins show the presence of a total of 14 subgroups, which can be divided into seven alpha- and seven beta-type groups. Including the Pre5 and Pre6 proteins, every subgroup contains a single yeast member. We anticipate that the 14 genes encoding subunits of the yeast proteasome represent the complete set of proteasomal genes of this organism. The ancestral archaebacterial proteasome is composed of four stacks of rings, the two outer rings containing seven identical alpha-subunits and the inner rings containing seven identical beta-subunits. We speculate that, in analogy to the archaebacterial proteasome, every eukaryotic proteasome is made of two halves of 14 distinct subunits, each half consisting of seven different alpha-type and 7 different beta-type subunits. In higher eukaryotes, subunit isoforms may contribute to variability in the subunit composition of the 20S proteasome allowing functional modulations.