Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyl-transferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA ( tRNAPyl) that presumably recognizes this UAG codon. It was reported that Lys- tRNAPylcan be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462, whereas a later article showed that Lys- tRNAPylis synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl- tRNAPylformation was presumed to result from subsequent modification of lysine attached to tRNAPyl. To investigate whether pyrrolysine can be directly attached to tRNAPylwe chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNAPyl; lysine and tRNALysare not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNAPylwith lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl. This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.