tRNA(His) guanylyltransferase (Thg1) post-transcriptionally adds a G (position -1) to the 5'-terminus of tRNA(His). The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNA(Pyl), the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp insertion yields active MaThg1 variants in M. acetivorans and equal amounts of full-length protein. These data suggest that Pyl insertion is akin to natural suppression and unlike the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play an ordinary role in proteins.