We have characterized a 17-residue peptide, MgtL, which is translated specifically in high Mg(2+) from an open reading frame (ORF) embedded in the Mg(2+) riboswitch domain, previously identified in the 5' leader region of Mg(2+) transporter gene mgtA in Salmonella. We demonstrate that mgtL translation is required to prematurely terminate mgtA transcription. Abrogation of mgtL translation by mutation of its start codon results in transcription of the mgtA-coding region in high Mg(2+), suggesting that ribosome stalling is not required for preventing premature transcription termination. Consistently, the Mg(2+) riboswitch responds to cytoplasmic Mg(2+), but not to proline or arginine, both repeatedly present in the MgtL sequence, to mediate mgtL translation-coupled regulation. RNA structural probing and nucleotide substitution analysis show that the riboswitch loop A region alters base pairing in response to Mg(2+), and favours stem-loop A1 in high Mg(2+), subsequently opening the ribosome-binding sequence for mgtL translation. Presumably, mgtL ORF directs translation to localize a ribosome in cis to act on downstream RNA in a manner similar to some upstream ORFs in prokaryotes and eukaryotes.