Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNA(Sec) by O-phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion of O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNA(Sec). Although SerRS recognizes both tRNA(Sec) and tRNA(Ser) species, PSTK must discriminate Ser-tRNA(Sec) from Ser-tRNA(Ser). Based on a comparison of the sequences and secondary structures of archaeal tRNA(Sec) and tRNA(Ser), we introduced mutations into Methanococcus maripaludis tRNA(Sec) to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNA(Sec) from tRNA(Ser). Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNA(Sec) were crucial for discrimination from tRNA(Ser). Furthermore, the A5-U68 base pair in tRNA(Ser) has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNA(Ser)(UGA) scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNA(Sec), whereas tRNA(Ser) did not. Additionally, the seryl moiety of Ser-tRNA(Sec) is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNA(Sec).