Aminoglycoside antibiotics bind directly to 16S rRNA in the 30S subunit of bacterial ribosomes and decrease the fidelity of translation. Aminoglycoside antibiotics remain important therapeutic agents and represent the archetype for RNA-targeted antibiotics. This chapter presents an overview of the investigations of how aminoglycoside antibiotics bind to rRNA and the insights these studies have provided into the decoding process. Throughout the chapter, the term “aminoglycoside” will implicitly refer to this subclass. It is within this conserved decoding region RNA that aminoglycoside antibiotics bind. The chemical groups that are common among aminoglycoside antibiotics direct specific interaction with the RNA. The major sequence difference between all prokaryotic ribosomes and all eukaryotic ribosomes in the aminoglycoside binding site is an A1408-toG1408 change. However, only low-level resistance was observed to G418 and paromomycin, which are the most effective aminoglycosides against eukaryotic organisms. The major mechanism of aminoglycoside resistance is enzymatic modification of the drug. Molecular contacts between A1492 and A1493 and mRNA during decoding may also explain the miscoding induced by aminoglycoside antibiotics. The work on aminoglycoside antibiotics has revealed the details of how aminoglycosides bind to the ribosome, how resistance occurs, and how selectivity for prokaryotes is achieved. Only the combination of structure determination, biochemical, and biophysical approaches provides true insights into the workings of the ribosome. The use of a small oligonucleotide was essential for this work.