Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes. We isolated a Tn917-insertional mutant of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level. Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain. Genetic analysis of the Tn917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred immediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expression whereas it had activated expression of mdrL. orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B. subtilis (21-24% identity), two members of the multidrug resistance efflux pump family. Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.