Thirty-three proteins have been isolated from the 50S ribosomal subunit of Escherichia coli by a technique based solely upon ion-exchange chromatography. The procedure can be adapted to a wide range of sample sizes, requires no prefractionation of the subunit proteins, and employs readily regenerated chromatographic media. The molecular weights, purity, and immunological properties of the individual proteins have been characterized. More than 20 of the proteins were judged to be at least 95% pure by electrophoretic analysis; the remaining proteins were generally over 90% pure. Methods for the immunological identification of small amounts of ribosomal proteins are described.