Pyrrolysine is a naturally occurring genetically coded amino acid. It is coded by the amber stop codon UAG. Structurally it is N6-[(2R,3R)-3-methyl-3,4-dihydro-2H-pyrrol-2-ylcarbonyl]-L-lysine.

Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with pyrrolysine. Homologs of pylS and pylT are found in a Gram-positive bacterium, Desulfitobacterium halfniense, although the function of these putative genes in this organism is unknown. Recently, it has been shown that the tRNA(CUA) can be charged with lysine in vitro by the concerted action of the M. barkeri Class I and Class II Lysyl-tRNA synthetases. Charging a tRNA(CUA) with lysine was originally hypothesized to be the first step in translating UAG amber codons as pyrrolysine in certain methanogens . The current model based on in vitro and in vivo data favors direct charging of pyrrolysine on to the tRNA(CUA) by the protein product of the pylS gene. This makes Pyrrolysine the 22nd genetically encoded natural amino acid. The mechanism of encoding makes it the 21st natural directly encoded amino acid.

Pyrrolysine is an amino acid used by some archaea in enzymes that are part of their methane-producing metabolism. It is coded for by a UAG codon, which is normally a stop codon named "amber" but whose meaning might possibly be modified by the presence of a specific downstream sequence, named PYLIS, which forms a stem-loop in the mRNA, forcing the incorporation of pyrrolysine instead of terminating translation.

See also selenocysteine, the 21st genetically encoded natural amino acid.