Staphylococcus aureus releases a large number of exoproteins, including membrane-active proteins and toxins with superantigenic activity involved in pathogenicity. However, the export pathways of exoproteins in S. aureus have not been reported. We analyzed the function of the staphylococcal twin-arginine translocation (Tat) pathway homologue, the presence of which was recently discovered according to the genome database. The amino-acid sequences of the Tat homologues of S. aureus do not have a high similarity with those of Escherichia coli and other bacteria. Constructed tatC-deficient mutants from distinct parent strains showed the same patterns of exoproteins compared with those of parent strains on two-dementional gel electrophoresis, and the amounts of secreted staphylococcal enterotoxins and toxic shock syndrome toxin-1, of which signal peptides have some features often seen in signal sequences of Tat-dependent proteins, did not change with Western blotting analyses. Therefore, it seems that the Tat pathway does not play a major role in the secretion system of S. aureus, but other export pathways may play an important role in toxin secretion. This is the first experimental report showing the influence of the Tat pathway on the secretion of S. aureus.