As it has become increasingly clear that the RNA components of the ribosome are central to its function, the in vitro analysis of mutations in the ribosomal RNAs has become an important tool for understanding the molecular details of ribosome function. However, the frequent dominant lethal phenotypes of mutations at interesting rRNA residues has long presented a hurdle to this analysis, as their lethality has rendered it impossible to generate pure populations of in vivo-derived ribosomes for study. We present here the details of a method for affinity purification of ribosomes bearing any mutation in the 16S or 23S rRNA and demonstrate that ribosomes purified using this technology are highly active in the several steps of translation we have examined.