The 2-deoxystreptamine-containing aminoglycosides are an important class of clinically valuable antibiotics. A deep understanding of the biosynthesis of these natural products is required to enable efforts to rationally manipulate and engineer the biological production of novel aminoglycosides. This review discusses the development of our biosynthetic knowledge over the past half-century, with emphasis on the relatively recent contributions of molecular biology to the elucidation of these biosynthetic pathways.