The minimal inhibitory concentrations (MICs) of ampicillin, tetracycline, and metronidazole for 71 strains of Haemophilus vaginalis (Corynebacterium vaginale) were compared by use of an agar-dilution method and an inoculum of 10(6) organisms/ml. All strains were sensitive to 1 microgram of ampicillin/ml, 70% to 4 micrograms of tetracycline/ml, and only 13% of the strains to 8 micrograms of metronidazole/ml. Under anaerobic conditions the susceptibility to metronidazole increased markedly, and 48% of the strains were inhibited by 8 micrograms/ml. In determinations of MICs in broth cultures, reduction of the inoculum size to 10(4) organisms/ml increased susceptibilities to metronidazole and tetracycline, whereas incubation of 48 hr instead of 24 hr decreased susceptibilities to these two drugs. Minimal bactericidal concentrations (MBCs) were generally two- to fourfold greater than the MICs for the three drugs. The results demonstrate that anaerobic conditions, inoculum size, and duration of incubation influence the susceptibility of H. vaginalis to antibiotics in vitro.