A number of archaeal organisms generate Cys-tRNA Cys in a two-step pathway, first charging phosphoserine (Sep) onto tRNA Cys and subsequently converting it to Cys-tRNA Cys . We have determined, at 3.2-Å resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, α 4 synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (αβ) 2 phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.