Single-copy integration vectors suitable for cloning in Staphylococcus aureus have been constructed. Their construction was based on the site-specific recombination system of staphylococcal phage, L54a. The vectors are capable of autonomous replication in Escherichia coli, but they are not endowed with a replication function in S. aureus. As a consequence, establishment of these vectors in S. aureus can only be achieved by the integration system of the phage. Once integrated into the chromosome, the vectors, or their derivatives, were stably inherited even without selective pressure. Because such a vector exists in an integrated form in S. aureus, the gene dosage of the DNA cloned in the vector matches that of the chromosome.