Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA(Pyl) and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA(Sec) and Pyl-tRNA(Pyl) formation as well as the distribution of the Pyl-decoding trait.