Antisense technology has been widely used to regulate gene expression. A tetracycline (tet)-regulated antisense-RNA-expressing system has been developed and used to downregulate chromosomally derived genes expressed in Staphylococcus aureus. This downregulation subsequently provides an evaluation of the virulence factor and drug targets. The regulated antisense RNA library allows for genomewide analyses of the functions of staphylococcal gene products for growth in culture and survival during infection. Moreover, this antisense RNA technology may provide a key tool to identify mechanisms of novel antibacterial compound action.