The ability of the foodborne pathogen Listeria monocytogenes to develop biofilm in food-processing environment is a major concern for the food safety, because biofilms allow bacteria to better resist environmental stresses. PrfA is a key transcriptional activator that positively regulates most of the known listerial virulence gene expression. In order to explore the role of PrfA on Listeria biofilm development, we compared the abilities of biofilm formation by L. monocytogenes wild type strains (EGD and EGDe) and their prfA deletion mutants (EGDa dagger prfA and EGDea dagger prfA), nonpathogenic Listeria innocua, as well as the recombinant strains that express constitutively active mutant PrfA (PrfA*) in L. innocua (LI-pERL3-prfA*) and in EGDea dagger prfA (EGDea dagger prfA-pERL3-prfA*) at 37A degrees C in brain heart infusion (BHI) medium using the polyvinyl chloride (PVC) microtiter plate assay and microscopic examination. Our results showed that the wild types of L. monocytogenes had strong abilities to develop biofilm with meshwork of bacterial aggregates, while biofilm with sparse small clumps were observed in L. innocua. The biofilm production of strains EGDa dagger prfA and EGDea dagger prfA that lack funtional PrfA was reduced and could be recovered by the introduction of the PrfA*, however, the PrfA* had no impact on the biofilm forming ability of L. innocua. Our results suggest that PrfA plays a significant role in biofilm formation in L. monocytogenes but not in L. innocua, thus may reflect differences in the molecular mechanisms of biofilm formation by these two closely related species.