Publisher Summary The Escherichia coli (E. Coli) ribosome is the best-characterized system in which translation has been investigated at the molecular level. The E. coli ribosome is a (70s) complex of RNA and protein composed of two subunits, the large (50s) subunit and the small (30s) subunit. This chapter reviews the structure and the function of 16s ribosomal RNA in E. coli. The chapter investigates the effects of mutations introduced into rRNA genes. Expression of rRNA genes is essential and there are seven copies of the rRNA genes in the E. coli genome. Mutational analysis of 16s ribosomal RNA structure and function is a powerful approach to the study of the role of this RNA in the process of translation. There is also great promise in two novel genetic approaches to the study of 16s rRNA—inactivation of as many as four chromosomal rrn operons in E. coli by insertion–deletion mutagenesis using antibiotic resistance cassettes and the introduction of antibiotic resistance mutations into the single chromosomal rRNA operon of Halobacterium halobium.