Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).