Studies of the Staphylococcus aureus LytSR two-component regulatory system have led to the identification of the cid and lrg operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The cid gene products enhance murein hydrolase activity and antibiotic tolerance whereas the lrg gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a lytS knockout mutant was generated from a clinical S. aureus isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for lrgAB expression. Furthermore, the lytS mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the lrgAB operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in S. aureus biofilm development, likely as a result of its direct influence on lrgAB expression.