Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report, we demonstrate that L27 inStaphylococcus aureusand other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram-negative bacteriumEscherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome inS. aureusand other related bacteria that differ substantially from that of theE. coliribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway.