Reverse-phase high-performance liquid chromatography has been used to fractionate ribosomal proteins from Escherichia coli and rabbit reticulocytes. Different column packing materials and solvent systems were compared for their effectiveness with bacterial proteins. A large-pore (300 A) short alkyl chain support (Altex RPSC) in conjunction with a triethylamine phosphate (pH 2.2)/acetonitrile solvent system was particularly effective and separated mixtures of total protein from each ribosomal subunit into a number of peaks approaching the actual number of proteins present. For example, with the use of the Altex RPSC column, the 21 proteins of 30S subunits were resolved into 18 distinct peaks, and the 33 proteins of the 50S subunits were resolved into 28 peaks. Overall recovery varied from 75% to 90% in different experiments. The composition of each peak was established by two-dimensional gel electrophoresis. Relatively acidic proteins, for example, S1 and L7/L12 of Escherichia coli, were bound more tightly to the column and recovered in lower yields than the other more basic proteins. Proteins that were incompletely resolved in a single step could be obtained in pure form by rechromatography on the same column with an altered gradient or with a different type of reverse-phase packing material. Ribosomal proteins from rabbit reticulocytes were also separated with good resolution and yield by using the RPSC column.