Ribosomal protein L4 is a regulator of protein synthesis in the Escherichia coli S10 operon, which contains genes of 11 ribosomal proteins. In this work, we have investigated regulatory functions of ribosomal protein L4 of the thermophilic archaea Methanococcus jannaschii. The S10-like operon from M. jannaschii encodes not 11, but only five ribosomal proteins (L3, L4, L23, L2, S19), and the first protein is L3 instead of S10. We have shown that MjaL4 and its mutant form lacking an elongated loop specifically inhibit expression of the first gene of the S10-like operon from the same organism in a coupled transcription-translation system in vitro. By deletion analysis, an L4-binding regulatory site has been found on MjaL3 mRNA, and a fragment of mRNA with length of 40 nucleotides has been prepared that is necessary and sufficient for the specific interaction with the MjaL4 protein.