Pus10, a recently identified pseudouridine (Psi) synthase, does not belong to any of the five commonly identified families of Psi synthases. Pyrococcus furiosus Pus10 has been shown to produce Psi55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Psi55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of Psi54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Psi54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA Psi54 and Psi55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Psi54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54 x A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Psi55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Psi54 synthase activity.