Staphylococcus aureus isolates (N = 40) from bovine mastitis were characterized by random amplified polymorphic DNA-PCR (RAPD-PCR), ribotyping and biotyping. The isolates were collected in the veterinary surveillance area of the Ambulatory Clinic, Faculty of Veterinary Medicine, University of Helsinki from 20 quarters during the acute phase of infection and from the same quarters 3 weeks after cessation of therapy. The aim of the study was to compare the S. aureus isolates taken from the same quarter at different times to verify persistence of virulent strains in infected quarters and to compare the discriminatory power of the diagnostic methods. Using all methods (except for a commercial diagnostic test), the paired isolates of S. aureus were identical. Results suggest that the chronic nature of S. aureus infections was due to the persistence of the original infective strain. More laborious ribotyping and the more convenient RAPD-PCR method produced identical results. The molecular methods differentiated the 40 isolates into 6 distinct genotypes. Biotyping produced partially identical results to RAPD and ribotyping. A commercial diagnostic test system identified only 3 S. aureus biotypes.