A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms. In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N ϵ, N ϵ-dimethyllysine, N ϵ, N ϵ, N ϵ-trimethyllysine, N G, N G-dimethylarginine (or N G-methylarginine), and N G, N′ G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N ϵ-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds. The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N G, N G-dimethylarginine (or N G-methylarginine), and smaller amounts of N G, N′ G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N ϵ, N ϵ, N ϵ-trimethyllysine and smaller amounts of N ϵ-methyllysine, N ϵ, N ϵ-dimethyllysine, and N G, N G-dimethylarginine. These subunits also contained an as yet unidentified methylated compound These results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N ϵ, N ϵ, N ϵ-trimethyllysine, with the remainder distributed about equally between N ϵ-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N ϵ-methyllysine was apparently in the small acidic proteins, L7 and L12.