The two pathogenic Listeria species, L. ivanovii and L. monocytogenes, can be differentiated biochemically and show different host ranges. Virulence of L. monocytogenes is dependent on the integrity of prfA which positively and co-ordinately regulates transcription of several virulence genes. Until now, a prfA homologue had not been identified in L. ivanovii. We have now cloned a chromosomal region from L. ivanovii comprising two genes with high homology to the plcA and prfA genes from L. monocytogenes. Distal from prfA, an open reading frame highly homologous to a phosphoribosyl pyrophosphate synthetase gene (prs) was newly identified, defining the border of the virulence gene cluster. Transcription of the gene for ivanolysin O and expression of other genes of the virulence gene cluster in L. ivanovii were dependent on PrfA. The pattern of PrfA-dependent proteins (PdPs) expressed in L. ivanovii was similar, but not identical to that of L. monocytogenes. The PrfA proteins, as predicted from nucleotide sequences of both pathogenic Listeria species, are very similar and show significant homology to the Crp-Fnr family of global transcription regulators.