The Enterococcus faecalis two-component signal transduction system CroRS, also referred as the RR-HK05 pair, is required for intrinsic β-lactam resistance (Y. R. Comenge, R. Quintiliani, Jr., L. Li, L. Dubost, J. P. Brouard, J. E. Hugonnet, and M. Arthur, J. Bacteriol. 185:7184-7192, 2003) and is also suspected to be involved in the expression of salB (previously referred to as sagA), a gene important for resistance to environmental stress and cell morphology (Y. Le Breton, G. Boël, A. Benachour, H. Prévost, Y. Auffray, and A. Rincé, Environ. Microbiol. 5:329-337, 2003). In this report, we provide genetic and biochemical evidence that salB encodes a secreted protein that is expressed from a monocistronic stress-inducible operon. Consistent with CroR being a direct transcriptional activator of the salB expression, CroR was found to bind to the salB promoter region in electrophoretic mobility shift assays. Interestingly, we provide evidence that SalB does not play a role in the intrinsic β-lactam resistance associated with CroRS. We also show that the CroRS system is able to regulate its own expression. The sequence of the CroRS binding site in the salB and croR promoter regions was determined using DNase I footprinting assays.