Staphylococcus epidermidis biofilm formation, the major pathogenic factor in medical device-associated infections, is regulated by a coplex regulatory network. By transposon mutagenesis in S. epidermidis 1457, a biofilm-negative mutant (M12) was isolated in which a new regulatory gene locus is inactivated. Primer extension analysis of a 0.95 kb transcript inactivated in M12 revealed a perfect consensus sequence of σ^B dependent promoters which regulates the expression of the downstream genes barA and barB (biofilm associated regulator A and B). We constructed knock out mutants of these genes by allelic gene replacement using Gateway Technology. Deletions of the respective genes were confirmed by western blot using antisera raised against the purified BarA and BarB proteins. The barA mutant shows reduced biofilm formation ability, whereas the barB and barAB mutants were biofilm-negative, the growth rate of all mutants was not significantly affected. Additionally, it could be demonstrated that PIA synthesis required for biofilm formation in S. epidermidis 1457 was drastically reduced (oder abolished?). In contrast to σ^B mutants PIA synthesis and biofilm formation could not be restored by adding Ethanol to the growth medium. Transcriptional analysis of icaA and icaR revealed that icaADBC transcription is drastically reduced in 1457barAB under all tested conditions, whereas there was no influence on icaR transcription. Indicating that the corresponding gene products of barA and barB represent regulators of icaADBC transcription functioning independently of the negative regulator IcaR. In the future we would like to characterize possible protein-protein interactions as well as protein-DNA interactions responsible for regulation of the expression of the icaADBC locus. However, this regulation is still a matter of debatte and needs to be further elucidated.