A total of 56 strains of Haemophilus vaginalis were tested for their in vitro susceptibility to 21 antimicrobial agents by an agar dilution method. All strains were inhibited by 1 mug or less of penicillin, ampicillin, carbenicillin, and vancomycin per ml. The cephalosporins were less active; 4 mug of cefazolin per ml, 16 mug of cephalothin per ml, or 128 mug of cephalexin per ml was required to inhibit all strains. Kanamycin, gentamicin, tobramycin, and neomycin were relatively inactive against H. vaginalis. All strains were inhibited by 4 mug of streptomycin per ml and 2 mug of chloramphenicol per ml. Only 57% of the strains were inhibited by 4 mug of tetracycline per ml, whereas 43% were inhibited by 16 to 64 mug/ml. The combination sulfamethoxazole/trimethoprim was relatively inactive against H. vaginalis. All strains tested exhibited minimal inhibitory concentrations of >/=128 mug when tested against colistin, nalidixic acid, and sulfadiazine. Erythromycin and clindamycin were the most active of the antibiotics tested; for all strains the minimal inhibitory concentrations were </=0.06 mug/ml.