The aroA gene of Staphylococcus aureus 8325-4 was cloned. Sequence analysis and the phenotype of directed plasmid insertions 5' to aroA suggest that aroA is located in an operon and that it maps 3' to the aroC and aroB genes. A revised consensus sequence for the aroA gene product EPSP synthase binding site for its substrate (phosphoenolpyruvate) and an inhibitor (glyphosate) is proposed. An aroA insertion mutant isolated by allelic replacement was employed in genetic mapping experiments which demonstrated the gene order thy aroA tyrB in SmaI fragment A of the S. aureus 8325-4 chromosome. The aroA::Tcr mutant required aromatic amino acids but remained independent of p-aminobenzoic acid (PAB). This could be due to the insertion being located close to the 5' end of the gene, allowing expression of a truncated protein. The PAB independence may explain the finding that the mutant was not attenuated in mouse infection experiments. It was not possible to isolate a null mutant in aroA.