We optimized a previously established two-plasmid system for the identification of Staphylococcus aureus promoters that are recognized by the alternative transcription factor sigma(B). The method allowed the identification of 18 S. aureus sigma(B)-dependent promoters, 12 of which are reported here for the first time to be sigma(B)-dependent. S1-nuclease mapping of the respective transcriptional start points revealed that all the promoters contained sequences exhibiting high similarity to the consensus sequence of Bacillus subtilis sigma(B)-dependent promoters. The promoters governed expression of genes encoding proteins proposed to be involved in various cellular functions, including the stress response genes and virulence-associated clfA gene for fibrinogen-binding clumping factor. Comparison of the nucleotide sequences upstream of the identified transcription start points identified a sigma(B) consensus promoter (GttTaa-N(12-15)-gGGTAt) that is highly homologous to that of sigma(B) of B. subtilis.