Primers were designed for polymerase chain reaction (PCR)-amplification of a genomic sequence specific to Staphylococcus aureus strains. The sequence corresponds to a part of the 44-kb Smal fragment (fragment L on the S. aureus NCTC 8325 restriction map) which was found to be common to strains of the S. aureus species (Pantucek et al., 1996, International journal of Systematic Bacteriology, 46: 216-222). The labelled 44-kb Smal restriction fragment derived from S. aureus NCTC 8325-4 was hybridized to the EcoRI restriction patterns of genomic DNA from 13 strains representing different macrorestriction types of S. aureus subsp. aureus. This made it possible to reveal the 2052 bp EcoRI restriction subfragment and to demonstrate its presence in all the tested strains. From the sequence of this subfragment, primers were designed by means of which the 826 bp amplicons were obtained in all 216 tested strains of S. aureus. No hybridization and PCR-products were observed in 40 collection strains of other staphylococcal species and subspecies as well as in 45 clinical strains of coagulase-negative staphylococci. These results lead us to the conclusion that the use of the above primers makes it possible to identify rapidly and reliably S. aureus strains of various provenance and different genotypes. (C) 2001 Academic Press.