SummaryAll known virulence genes of Listeria monocytogenes are under positive regulation by the transcription factor PrfA. Previous work employing the L. monocytogenes strain NCTC7973 suggested that the disaccharide cellobiose might serve as a specific 'signature molecule' which functions to prevent activation of the PrfAcontrolled regulon in a soil environment. We have examined three other L. monocytogenes strains, 10403S, LO28 and EGD, all commonly regarded as wild-type isolates, and find that NCTC7973 is anomalous with respect to the effect of carbohydrates on the expression of PrfA-controlled gene expression. In the case of 10403S, LO28 and EGD, several other readily metabolized mono-and disaccharides are as effective as cellobiose in repressing expression of the PrfA-controlled gene hly, indicating that the cellobiose effect is not specific, and suggesting that NCTC7973 may be a partially deregulated variant. Moreover, concentrations of cellobiose and other sugars required for repression of hly expression (> 1 mM) were found to significantly enhance growth of L. monocytogenes cultures, suggesting that the repression phenomenon probably results from a metabolic effect of sugar utilization rather than a signal-sensing response. Thus the previously reported cellobiose effect may reflect an aspect of a more global mechanism of catabolite repression in L. monocytogenes. Although cellobiose represses expression of hly and plcA at the level of transcript accumulation, quantitative Western blot analysis indicates that cellobiose has no effect on PrfA levels. These results are consistent with a model in which PrfA activity is controlled by interaction with a hypothetical cofactor, the synthesis or depletion of which is responsive to the presence of readily metabolized carbohydrates.