Chemiluminescent DNA probes (AccuProbe, species specific; and FlashTrack, bacterial generic) were used to determine oxacillin resistance in Staphylococcus aureus. Ribosomal RNA was measured at designated intervals in the presence and absence of antibiotic. A total of 48 (AccuProbe assay) and 24 (FlashTrack) S. aureus isolates with known oxacillin susceptibility patterns were inoculated into Bactec 6A bottles both with and without 4 micrograms/ml oxacillin and incubated at 35 degrees C for 4 h. Aliquots were removed at 0 and 4 h, and pellets of bacteria were obtained via selective centrifugation. Probe assay counts (relative light units, RLUs) were performed. Of 21 oxacillin-resistant S. aureus (ORSA) strains, 20 showed a > 5-fold RLU increase during the incubation period (Accu-Probe assay): 25 of 27 oxacillin-susceptible strains demonstrated a < or = 4-fold increase. AccuProbe test sensitivity and specificity were 95% and 92%, respectively. With the generic FlashTrack probe assay, all nine ORSA isolates showed a > or = 4- to 10-fold increase in RLUs, and all 15 oxacillin-susceptible strains showed a < or = 4-fold increase in RLUs during the 4-h incubation. The FlashTrack test sensitivity and specificity were both 100%. Probe assays were completed within 5 h. This study suggests that rapid and reliable determination of oxacillin resistance in S. aureus clinical isolates can be accomplished using commercially available DNA probes.