Aminoacyl tRNA synthetases are divided into two unrelated classes of ten enzymes each. Members from each class specifically aminoacylate small RNA oligonucleotides lacking anticodon sequences. Duplex structures with only four base pairs stabilized by RNA tetraloop motifs are active. Atomic groups on bases and ribose 2'-hydroxyl groups in the RNA minor groove make functional contacts that are essential for aminoacylation and provide the high specificity. A system for specific aminoacylation of small RNA oligonucleotides that is based on sequences proximal to the amino acid attachment site may reflect the small sizes of early synthetases.