A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5′ terminus of the genearlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlSforms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, thearlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locusarlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence ofS. aureus.