An antimicrobial susceptibility test, a tow-tube broth dilution and disk elution method for Ureaplasma urealyticum, was modified to incorporate some of the standard procedures followed in traditional antimicrobial testing. The susceptibility pattern of the species was reevaluated by determining the effect of various antimicrobial agents on 21 vaginal isolates. All isolates were inhibited by tetracycline congeners (1 to 6 micrograms/ml) and killed by methenamine mandelate (0.6 mg/ml). All but one isolates were inhibited by erythromycin (0.4 to 3 micrograms/ml). Only eight isolates were inhibited by nalidixic acid (1 to 6 micrograms/ml), and seven were inhibited by nitrofurantoin (20 to 60 micrograms/ml), whereas all isolates were resistant to rifampin (1 microgram/ml) and trimethoprim-sulfamethoxazole (5 micrograms/ml). The in vitro technique described can readily be performed on individual patient isolates before the initiation of antimicrobial therapy.