The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility-shift assays (EMSAs) using cell-free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA-specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA-specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain-heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA-free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA-free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA-containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.