The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an l-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the -35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI'-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352'-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.