The general pathway involving adenosine triphosphate (ATP)-dependent proteases and ATP-independent peptidases during cytosolic protein degradation is conserved, with differences in the enzymes utilized, in organisms from different kingdoms. Lon and caseinolytic protease (Clp) are key enzymes responsible for the ATP-dependent degradation of cytosolic proteins in Escherichia coli. Orthologs of E. coli Lon and Clp were searched for, followed by multiple sequence alignment of active site residues, in genomes from seventeen organisms, including representatives from eubacteria, archaea, and eukaryotes. Lon orthologs, unlike ClpP and ClpQ, are present in most organisms studied. The roles of these proteases as essential enzymes and in the virulence of some organisms are discussed.