Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing Eco RI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n = 12), hybridized in a homogeneously positive fashion (n = 13) or hybridized only with their source strains (n = 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.