Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase. RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.