The involvement of defined regions of Escherichia coli 16S rRNA in the fidelity of decoding has been examined by analyzing the effects of rRNA mutations on misreading errors at the ribosomal A and P sites. Mutations in the 1400-1500 region, the 530 loop and in the 1050/1200 region (helix 34) all caused readthrough of stop codons and frameshifting during elongation and stimulated initiation from non-AUG codons at the initiation of protein synthesis. These results indicate the involvement of all three regions of 16S rRNA in decoding functions at both the A and P sites. The functional similarity of all three mutant classes are consistent with close physical proximity of the 1400- 1500 region, the 530 loop and helix 34 and suggest that all three regions of rRNA comprise a decoding domain in the ribosome.