The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution. Molecular & Cellular Proteomics 9: 1774 -1783, 2010.Ribosomes universally translate the genetic code into proteins. They consist of two asymmetric subunits between which mRNA is decoded and amino acids are added to a growing peptide chain. On the large subunit there is a noticeable protrusion, observable by electron microscopy, known as the stalk complex (also denoted as L8) (1). This complex is involved in the binding and orientation of translation factors and exists with variable composition throughout all three domains of life. In bacteria we and others have shown previously that it is composed of either two or three dimers of the protein L12 (termed L7 when N-acetylated) attached to a single copy of the scaffolding protein L10 (2, 3). These assemblies of stalk proteins, either L10(L7/L12) 4 or L10(L7/L12) 6 , are referred to as pentameric or heptameric stalk complexes hereafter. In eukaryotes there is an identical arrangement for the stalk complex but of unrelated proteins with no sequence homology to L10/L12. In this case P0 is the L10 equivalent scaffolding protein, and two different but related proteins (P1 and P2) take the place of L12 (nomenclature according to Ref. 4). In plants P3 occurs in addition to P1 and P2 (5). The Pproteins are named after their propensity for phosphorylation when attached to the ribosome. In yeast, but not in higher eukaryotes, P1 and P2 have both evolved into two ␣ and  proteins (6). In archaea the stalk complex constituents, although named L10 and L12, share sequence homology with the P-proteins (7). L12 and its P1/P2 counterparts are the only ribosomal proteins that have acidic pI values, do not interact directly with rRNA, and are present in multiple copies on the ribosome.We have shown previously that by applying a combination of MS and tandem MS approaches to intact MDa par...