The current novobiocin spectrum of antimicrobial activity was evaluated by testing 585 staphylococci (393 oxacillin-resistant) and 779 other bacterial strains. Novobiocin inhibited 98.5% of all staphylococcal isolates (MIC 90, 0.25 micrograms/ml) and a significant number of other Gram-positive species at less than or equal to 1 microgram/ml. Pathogenic Neisseria, Haemophilus influenzae, and Branhamella catarrhalis were also very susceptible to novobiocin, e.g., MIC 100, less than or equal to 1 microgram/ml. These results indicate that novobiocin may have a role in contemporary chemotherapy of oxacillin-resistant staphylococcal and other infections.