In eukaryotes, the proteasome represents the major system for controlled intracellular proteolysis. Its ubiquitin-independent counterpart in prokaryotes is structurally well characterized, but still little is known about its cellular targets and hence its physiological role (M. J. Pearce, P. Arora, R. A. Festa, S. M. Butler-Wu, R. S. Gokhale, and K. H. Darwin, EMBO J. 25:5423, 2006; S. Gandotra S, D. Schnap-pinger, M. Monteleone, W. Hillen, and S. Ehrt, Nature Med. 13:1515, 2008). From an evolutionary point of view, this compartmentalized protease is also of interest as a possible ancestor of the eukaryotic system (R. E. Valas and P. E. Bourne, J. Mol. Evol. 66:494, 2008). Although widespread in Archaea, occurrence of the 20S proteasome (28-subunit barrel-shaped proteolytic core) appeared to be confined to a single phylum of gram-positive bacteria, the Actinobacteria, including genera like Mycobacterium, Rhodococcus, and Streptomyces. Recently, this viewpoint needed adjustment upon proteogenomic identification in mine-drainage biofilm of actinomycete-like proteasomes from uncultivated Leptospirilla, acidophilic gram-negative bacteria of the Nitrospirae phylum (R. De Mot, Trends Microbiol. 15: 335, 2007). Now, genomic data for two cultivated soil isolates of the equally poorly characterized phylum Verrucomicrobia reveal that equivalents of the characteristic six-gene cluster previously identified in Leptospirillum (mpa-pafX-orfX-prcB-prcA-pafA) are present in the extremely acidophilic methanotroph ‘Methylacidiphilum infernorum’ V4 (P. F. Dunfield et al., Nature 450:879; GenBank CP000975) and the xylan-utilizing strain Ellin514 (P. Sangwan, S. Kovac, K. E. Davis, M. Sait, and P. H. Janssen, Appl. Environ. Microbiol. 71:8402, 2005; Gen-Bank ABOX010000075). The moderate level of homology between the respective verrucomicrobial orthologues (46–54 % amino acid identity) and significant difference in G + C-content of the gene clusters (10%) reflect the different subdivision affiliation of these strains within the phylum. Much like the two Leptospirillum proteasome gene clusters, the genome context of both verrucomicrobial clusters is not conserved and lacks synteny with flanking genes in actinomycete genomes.