Rifampin was studied for determination of its spectrum of activity against many bacteria of clinical importance. Most of the minimum inhibitory concentrations (MICs) were determined by agar dilution but some were determined by broth microdilution. Staphylococci were the most susceptibile, with mode MICs of 0.015 μg/ml, but most streptococcal strains, except Streptococcus faecalis, had mode MICs ⩽1 μg/ml. Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, and Listeria monocytogenes were susceptible and had mode MICs of 1, 0.25, 0.03, and ⩽ 0.12 μg/ ml, respectively. Legionella species had geometric mean MICs ranging from 0.027 to 0.25 μg/ml. The rapidly growing mycobacteria, Mycobacterium chelonei and Mycobacterium fortuitum, were resistant, with mode of >64 μg/ml. Enterobacteriaceae, Acinetobacter species, and Pseudomonas species had mode MICs ranging from 4 to 64 μg/ ml. Thus, the authors conclude that, on the basis of these in vitro data and an MIC breakpoint of ⩽2 μg/mI, gram-positive cocci (except for some enterococci), H. influenzae, N. gonorrhoeae, N. meningitidis, Legionella, and L. monocytogenes may be clinically susceptible to rifampin.